Cochleate compositions directed against expression of proteins

ABSTRACT

Disclosed herein are novel siRNA-cochleate and morpholino-cochleate compositions. Also disclosed are methods of making and using siRNA-cochleate and morpholino-cochleate compositions.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/822,235 filedApr. 9, 2004, which_claims the benefit of U.S. Provisional ApplicationNo. 60/461,483, filed Apr. 9, 2003; U.S. Provisional Application Ser.No. 60/463,076, filed Apr. 15, 2003; U.S. Provisional Application Ser.No. 60/502,557, filed Sep. 11, 2003; U.S. Provisional Application No.60/499,247 filed Aug. 28, 2003; U.S. Provisional Application No.60/532,755, filed Dec. 24, 2003. The entire contents of each of theaforementioned applications are hereby expressly incorporated herein byreference in their entireties.

BACKGROUND OF THE INVENTION

In diverse eukaryotes, double-stranded RNA (dsRNA) triggers thedestruction of mRNA sharing sequence with the double-strand (Hutvdgneret al. (2002) Curr. Opin. Genet. Dev. 12:225-232; Hannon (2002) Nature418:244-251). In animals and basal eukaryotes, this process is calledRNA interference (RNAi) (Fire et al. (1998) Nature 391:806-811). Thereis now wide agreement that RNAi is initiated by the conversion of dsRNAinto 21-23 nucleotide fragments by the multi-domain RNase III enzyme,Dicer (Bernstein et al. (2001) Nature 409:363-366; Billy et al. (2001)Proc. Natl. Acad. Sci. USA 98:14428-14433; Grishok et al. (2001) Cell106:23-34; Ketting et al. (2001) Genes Dev. 15:2654-2659; Knight et al.(2001) Science 293:2269-2271; and Martens et al. (2002) Cell13:445-453). These short RNAs are known as small interfering RNAs(siRNAs), and they direct the degradation of target RNAs complementaryto the siRNA sequence (Zamore et al. (2000) Cell 101:25-33; Elbashir etal. (2001) Nature 411:494-498; Elbashir et al. (2001) Genes Dev.15:188-200; Elbashir et al. (2001) EMBO J 20:6877-6888; Nykdnen et al.(2001) Cell 107:309-321; and Elbashir et al. (2002) Clin. Pharinacol.26:199-213).

siRNA molecules typically have 2- to 3-nucleotide 3′-overhanging ends,which permits them to be capable of interacting with an endonucleasecomplex, which results in a targeted mRNA cleavage. The potentialtherapeutic use of siRNA has been demonstrated in a number of systems.RNAi technology has been utilized to successfully target various genes,including HIV rev genes, CD4 and CD8 genes, and P53 genes (Lee, N. S. etal. (2002) Nature Biotechnol. 20: 500-505; Brummelkamp, T. R., et al.Science 2002. 296: 550-553.).

siRNAs have been used in a number of different experimental settings tosilence gene expression. For example, chemically synthesized or in vivotranscribed siRNAs have been transfected into cells, injected into mice,or introduced into plants (e.g. by a particle gun). Additionally, siRNAshave been expressed endogenously from siRNA expression vectors or PCRproducts in cells or in transgenic animals. Besides being utilized forgene silencing, siRNAs have been determined to play diverse biologicalfunctions in vivo. This includes roles that include antiviral defense,transposon silencing, gene regulation, centromeric silencing, andgenomic rearrangements. Such functional diversity has exemplified theimportance of siRNAs within cells and has also stirred interest in theirdetection across species and tissues. Gene Specific Silencing by RNAi,Tech Notes 10(1) from <<http://www.ambion.com/techlib/tn/101/7.html>>(visited Apr. 1, 2004). McManus M T and Sharp P A (2002) Gene silencingin mammals by small interfering RNAs. Nature Rev Genet 3: 737-747.Dillin A (2003) Proc Natl Acad Sci USA 100(11): 6289-6291. Tuschl T(2002) Nature Biotechnol 20: 446-448.

An obstacle to the realization of the full potential of gene therapy isthe development of safe and effective means for delivering siRNA tocells and organisms. The use of antisense oligonucleotides astherapeutic agents has also been widely investigated in the past fewyears. Gould-Fogerite et al. Cochleate Delivery Vehicles: Applicationsto Gene Therapy. Drug Delivery Technology, Vol 3:40-47, 2003. Parker etal. In Vivo and in vitro anti-proliferative effects of antisense IL-10Oligonucleotides in Antisense Technology, Part B, M. Ian Phillips, Ed.,Methods in Enzymology, Vol 314, pp 411-429, 1999; Mannino et al., NewGeneration Vaccines: “Antigen cochleate formulations for oral andsystemic vaccination,” p. 1-9 (Marcel Dekker, New York, 2^(nd) ed.1997); Brent et al., Neurosci 114(2): 275-278 (2002); Akhtar et al.,Nucleic Acids Res. 19:5551 (1991). Their efficacy is based on theirability to recognize their mRNA target in the cytoplasm and to blockgene expression by binding and inactivating selected RNA sequences.

While the potential of antisense is widely recognized, there arenumerous limitations to the use of antisense currently available. One ofthe key limiting aspects of this strategy is poor cell penetration.Akhtar et al., Nucleic Acids Res. 19:5551 (1991).

Morpholino oligonucleotides (also referred to herein as “morpholinos”)are oligonucleotides that include an antisense oligonucleotide andmorpholine backbone. These antisense morpholinos, typically 18-25nucleotides in length, can be designed to bind to a complementarysequence in a selected mRNA. The binding of the morpholino to the“target sequence” prevents translation of that specific mRNA, therebypreventing the protein product from being made. Morpholinos function byan RNase H-independent mechanism (i.e., a steric block mechanism asopposed to an RNase H-cleavage mechanism), and are soluble in aqueoussolutions, with most being freely soluble at mM concentrations(typically 10 mg/ml to over 100 mg/ml). Nasevicius et al., Nat. Genet26:216-220 (2000); Lewis et al., Development 128:3485-95 (2001);Mang'era et al., Eur. J. Nucl. Med. 28:1682-1689 (2001); Satou et al.,Genesis 30:103-06 (2001); Tawk et al., Genesis 32:27-31 (2002); Lebedevaet al., Annu. Rev. Pharmacol. Toxicol. 4:403-19 (2001).

Morpholinos have numerous, significant advantages over the alternativephosphorothioates, which have been documented with a number ofnon-antisense effects. Morpholinos generally are stable in cells becausetheir morpholine backbone is not recognized by nucleases. In addition,morpholinos are highly effective with predictable targeting, as comparedto other antisense molecules. Nasevicius et al., Nat. Genet 26:216-220(2000); Lewis et al., Development 128:3485-95 (2001); Mang'era et al.,Eur. J. Nucl. Med. 28:1682-1689 (2001); Satou et al., Genesis 30:103-06(2001); Tawk et al., Genesis 32:27-31 (2002); Lebedeva et al., Annu.Rev. Pharmacol. Toxicol. 4:403-19 (2001).

Key parameters for antisense inhibition by antisenseoligodeoxiribonucleotides are their intracellular delivery andconcentration. At the present time, it is believed that nakedoligonucleotides enter the cell via active processes of adsorptiveendocytosis and pinocytosis. However, the penetration of the endosomalbarrier is a pre-requisite event for antisense activity and the nakedantisense oligonucleotides do not appear to do this in great extent.Lebedeva et al., Annu. Rev. Pharmacol. Toxicol. 4:403-19 (2001); Weisset al., Neurochem. Int. 31:321-48 (1997). Although complexes ofantisense oligonucleotides with cationic liposomes, in some instances,have enhanced intracellular delivery, they have come with adisadvantage, cytotoxicity. Their utility in vitro and in vivo has alsobeen limited by their lack of stability in serum and their inflammatoryproperties.

Conventional methods for the delivery of morpholinos in vitro includescrape loading and the so-called “special delivery vehicles.” Scrapeloading entails adding oligonucleotides to adherent cells and scrapingthe cells from their plate, which disrupts the cell membrane temporarilyallowing the oligonucleotide to enter the cell cytoplasm. Scraping thecells causes damage to the membrane, thereby reducing the viability ofthe cell population and ultimately altering the cellular characteristicsof the remaining viable cells. Of the cells that do survive, not all mayhave received the morpholino. The second method, the “special deliveryvehicle” supplied with the morpholino, requires dramatic changes in pHthat result in very low efficacy. The low efficacy of the “specialdelivery vehicle” may be due to cytotoxicity or other changes to thecells.

The above methods are not translatable to in vivo delivery because theyinvolve compromise of the target cells and pH changes. Furthermore, anyin vivo delivery method or product must deliver the oligonucleotide tothe cytosol. Without delivery to the cytosol, oligonucleotides remaintrapped in the endosome/lysosome, or may be exocytosed.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods of deliveringsiRNA and morpholinos to cells and organisms employing cochleates. Alsoprovided are novel methods of forming cochleates and methods oftreatment and administration.

In one aspect, the invention provides an siRNA-cochleate compositionincluding a cochleate, and an siRNA associated with the cochleate. Incertain embodiments, the siRNA comprises at least one mismatch, at leastone substitution, and/or is about 21-23 nucleotides long.

In one embodiment, the siRNA mediates RNA interference against a targetmRNA. The target mRNA can be, e.g., an mRNA that expresses a proteinselected from the group consisting of: a cancer protein, a virusprotein, an HIV protein, a fungus protein, a bacterial protein, anabnormal cellular protein, a normal cellular protein. The compositioncan also include a second siRNA directed against a second target mRNA.In certain preferred embodiments, the composition includes a pluralityof siRNA against the same target mRNA.

In one embodiment, the cochleate includes a negatively charged lipidcomponent and a multivalent cation component. Additionally oralternatively, the siRNA is complexed with a transfection agent prior tocontacting the liposomes. The transfection agent can be a polycationictransfection agent, e.g., polyethylenimine (PEI) or a derivativethereof. The compositions of the invention can further include at leastone additional cargo moiety and/or at least one aggregation inhibitor.

In another aspect, the invention provides a method of administering ansiRNA to a host comprising: administering a biologically effectiveamount of an siRNA-cochleate composition to a host comprising acochleate and an siRNA associated with the cochleate. In one embodiment,the siRNA is delivered from the cochleate to a cell in the host. Inanother, the siRNA is delivered into a cytosol compartment of the cell.

In preferred embodiments, the siRNA mediates RNA interference against atarget mRNA in the host. In one embodiment, the target mRNA expressionin the host is reduced by at least about 50%. In other embodiments, thetarget protein synthesis in the host is reduced by at least about 10%,or at least about 50%. In certain embodiments, the host is a cell, acell culture, an organ, tissue, or an animal. The method may alsoinclude the step of examining the function of the target mRNA or proteinexpressed by the target mRNA in the host.

In yet another aspect, the invention provides a method of treating asubject having a disease or disorder associated with expression of atarget mRNA. The method includes administering to a subject atherapeutically effective amount of an siRNA-cochleate composition,including a cochleate and an siRNA against a target mRNA associated witha disease or disorder, such that the disease or disorder is treated.

In some embodiments, the disease or disorder is selected from the groupconsisting of: a neurological disorder associated with aberrant orunwanted gene expression, schizophrenia, obsessive compulsive disorder(OCD), depression, a bipolar disorder, Alzheimer's disease, Parkinson'sdisease, a lysosomal storage disease, Fabry's disease, Gaucher'sDisease, Type I Gaucher's Disease, Farber's disease, Niemann-Pickdisease (types A and B), globoid cell leukodystrophy (Krabbe's disease),metachromic leukodystrophy, multiple sulfatase deficiency, sulfatidaseactivator (sap-B) deficiency, sap-C deficiency, G_(M1)-gangliosidosis,Tay-Sachs disease, Tay-Sachs B1 variant, Tay-Sachs AB variant, AcidMaltase Deficiency, Mucopolysaccharidosis, Sandhoff's disease, a cancer,a cell proliferative disorder, a blood coagulation disorder,Dysfibrinogenaemia, hemophelia (A and B), dematological disorders,hyperlipidemia, hyperglycemia, hypercholesterolemia, obesity, acute andchronic leukemias and lymphomas, sarcomas, adenomas, a fungal infection,a bacterial infection, a viral infection, an autoimmune disorder,systemic lupus erythematosis, multiple sclerosis, myasthenia gravis,autoimmune hemolytic anemia, autoimmune thrombocytopenia, Grave'sdisease, allogenic transplant rejection, rheumatoid arthritis,ankylosing spondylitis, psoriasis, scleroderma, carcinomas, epithelialcancers, small cell lung cancer, non-small cell lung cancer, prostatecancer, breast cancer, pancreatic cancer, hepatocellular carcinoma,renal cell carcinoma, biliary cancer, colorectal cancer, ovarian cancer,uterine cancer, melanoma, cervical cancer, testicular cancer, esophagealcancer, gastric cancer, mesothelioma, glioma, glioblastoma, pituitaryadenomas, inflammatory diseases, osteoarthritis, atherosclerosis,inflammatory bowel diseases (Crohns and ulcerative colitis), uveitis,eczema, chronic rhinosinusitis, asthma, a hereditary disease, cysticfibrosis, and muscular dystrophy.

In yet another aspect, the invention provides a method of forming ansiRNA-cochleate composition that includes precipitating a liposome andan siRNA to form an siRNA-cochleate. In one embodiment, the methodincludes adjusting the pH of the siRNA and/or charging the base pairs ofthe siRNA.

In some embodiments, the siRNA is complexed with a transfection agentprior to precipitating. The transfection agent can be mixed with theliposomes prior to adding the siRNA. In one embodiment, the transfectionagent is PEI or a derivative thereof or other polyvalent cation.

The method can include using an elevated amount of calcium forprecipitating the liposome and the siRNA. Additionally or alternatively,the method can include the step of extruding the liposome with the siRNAprior to precipitation. In certain embodiments, the siRNA-liposome canbe prepared by adding a chelating agent to a cochleate to form aliposome in the presence of siRNA.

In yet another aspect, the invention provides a morpholino-cochleatecomposition that includes a cochleate; and a morpholino oligonucleotideassociated with the cochleate.

In one embodiment, the morpholino oligonucleotide is an antisensemorpholino oligonucleotide. The morpholino oligonucleotide can includeat least one mismatch and/or can be about 18-25 nucleotides long. Inpreferred embodiments, the morpholino oligonucleotide mediatesinhibition of translation of a target mRNA. In preferred embodiments,the morpholino oligonucleotide is also directed against the synthesis ofa protein.

In certain embodiments, the cochleate comprises a negatively chargedlipid component and a cation component, includes at least one additionalcargo moiety and/or includes at least one aggregation inhibitor. Thecomposition can also include a second morpholino oligonucleotidedirected against the synthesis of the protein or a second protein. Inother embodiments, the composition includes a plurality of morpholinosdirected against the same target mRNA.

In yet another aspect, the invention provides a method of administeringa morpholino oligonucleotide to a host. The method generally includesadministering a biologically effective amount of a morpholino-cochleatecomposition to the host comprising a cochleate and a morpholinooligonucleotide associated with the cochleate.

In one embodiment, the morpholino oligonucleotide is released from thecochleate into a cell in the host. In preferred embodiments, themorpholino oligonucleotide mediates inhibition of translation of atarget mRNA.

In certain embodiments, target mRNA expression in the host is reduced byat least about 50%, target protein synthesis in the host is reduced byat least about 10%, and/or target protein synthesis in the host isreduced by at least about 50%.

In certain embodiments, the host is a cell, a cell culture, an organ,tissue, or an animal, and/or the morpholino oligonucleotide is deliveredinto a cytosol compartment of a cell.

In yet another aspect, the invention provides a method of forming amorpholino-cochleate composition that includes the step of precipitatinga liposome and a morpholino to form a morpholino-cochleate.

The method can include the step of adjusting the pH of the morpholinoand/or charging the base pairs of the morpholino. The method can includeadjusting the pH of the morpholino to induce a charge in the morpholino.In one embodiment, the pH of the morpholino is between about 8.0 andabout 9.0.

The method can include using an elevated amount of calcium forprecipitating the liposome and the morpholino. The method can includeextruding the liposome prior to precipitation. In one embodiment, theliposome is prepared from addition of a chelating agent to a cochleateto form a liposome in the presence of morpholino. In some embodiments,the method includes adding at least one additional cargo moiety to themorpholino and the liposome prior to or after precipitating and/oradding an aggregation inhibitor to the morpholino and the liposome priorto or after precipitating.

In yet another aspect, the invention provides a method of treating asubject having a disease or disorder associated with expression of atarget mRNA. The method generally includes administering to a subject atherapeutically effective amount of an morpholino-cochleate composition,comprising a cochleate and an siRNA against a target mRNA associatedwith a disease or disorder, such that the disease or disorder istreated. The disease or disorder can be selected from the groupconsisting of: a neurological disorder associated with aberrant orunwanted gene expression, schizophrenia, obsessive compulsive disorder(OCD), depression, a bipolar disorder, Alzheimer's disease, Parkinson'sdisease, a lysosomal storage disease, Fabry's disease, Gaucher'sDisease, Type I Gaucher's Disease, Farber's disease, Niemann-Pickdisease (types A and B), globoid cell leukodystrophy (Krabbe's disease),metachromic leukodystrophy, multiple sulfatase deficiency, sulfatidaseactivator (sap-B) deficiency, sap-C deficiency, G_(M1)-gangliosidosis,Tay-Sachs disease, Tay-Sachs B1 variant, Tay-Sachs AB variant, AcidMaltase Deficiency, Mucopolysaccharidosis, Sandhoff's disease, a cancer,a cell proliferative disorder, a blood coagulation disorder,Dysfibrinogenaemia, hemophelia (A and B), dematological disorders,hyperlipidemia, hyperglycemia, hypercholesterolemia, obesity, acute andchronic leukemias and lymphomas, sarcomas, adenomas, a fungal infection,a bacterial infection, a viral infection, an autoimmune disorder,systemic lupus erythematosis, multiple sclerosis, myasthenia gravis,autoimmune hemolytic anemia, autoimmune thrombocytopenia, Grave'sdisease, allogenic transplant rejection, rheumatoid arthritis,ankylosing spondylitis, psoriasis, scleroderma, carcinomas, epithelialcancers, small cell lung cancer, non-small cell lung cancer, prostatecancer, breast cancer, pancreatic cancer, hepatocellular carcinoma,renal cell carcinoma, biliary cancer, colorectal cancer, ovarian cancer,uterine cancer, melanoma, cervical cancer, testicular cancer, esophagealcancer, gastric cancer, mesothelioma, glioma, glioblastoma, pituitaryadenomas, inflammatory diseases, osteoarthritis, atherosclerosis,inflammatory bowel diseases (Crohns and ulcerative colitis), uveitis,eczema, chronic rhinosinusitis, asthma, a hereditary disease, cysticfibrosis, and muscular dystrophy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D is photographs of cells treated with morpholino-cochleates.FIGS. 1A and 1B are photographs of rhodamine labeled cochleatesincubated with NGF differentiated PC12 cells at 3 hours and 12 hours,respectively, after cochleate introduction. As illuminated by thefluoresced rhodamine, the cochleates fuse with the outer membrane andform submembrane aggregates. FIGS. 1C (low power) and 1D (high power)are photographs of fluoresced rhodamine labeled cochleates containingfluorescein isothiocyanate (FITC) labeled morpholinos. FIGS. 1C-Dillustrate cochleates containing morpholinos, morpholinos that have beenreleased into the cytosol from unwrapped cochleates and the delivery ofFITC labeled anti-GAPDH Morpholino into the cytoplasm. Scale barsindicate 10 micrometers.

FIGS. 2A-B are photographs of X-Y RGCL LCSM computational slicesdemonstrating avid cochleate uptake by retinal ganglion cells in situ.

FIG. 3 includes two Western blots illustrating a decrease inglyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein at 18 and 24hours following treatment with a GAPDH antisense morpholino-cochleate(lower blot) while control cells receiving vehicle with cochleate alone(upper blot) showed no change in GAPDH protein levels.

FIGS. 4A-B are photographs of cells treated with morpholino-cochleatesdemonstrating delivery of the morpholinos into the cell cytosol andnucleus.

FIG. 5 is a graph summarizing in vivo antisense IL-10 experiments setforth Example 4.

FIG. 6 is a graph of absorption due to color development in the ELISAassay for surface erb B2 expression in SKOV cells for siRNA-cochleates,empty cochleates, and Lipofectamine formulated siRNA against erb B2expression.

FIG. 7 is a series of fluorescent confocal microscopy images of theSKOV3 cells 24 hours post-exposure to: empty cochleates (panel A), 1%rhodamine-labelled cochleates (panel B), anti-erb B2 siRNA-cochleates(panel C), and Cy5 labelled anti-erb B2 siRNA-cochleates (panel D),indicating successful delivery of siRNA to the cells and partialknockdown of cytoplasmic erb B2 expression.

FIG. 8 is a series of confocal microscopy images of SKOV3 cells 24 hourspost-exposure to: empty cochleates (panel A), 1% rhodamine-labelledcochleates (panel B), anti-erb B2 siRNA-cochleates (panel C), and Cy5labelled anti-erb B2 siRNA-cochleates (panel D), indicating successfuldelivery of siRNA to the cells and partial knockdown of cell surface erbB2 expression.

FIG. 9 is a graph indicating the partial knockdown effect of anti-erb B2siRNA-cochleates formulated with PEI, and washed to remove free siRNA,on SKOV3 cells.

FIG. 10 is a graph indicating indicated increased RNAi effect inencochleated siRNA versus unencochleated siRNA in SKOV3 cells.

DETAILED DESCRIPTION OF THE INVENTION

A novel approach to the delivery of siRNA and morpholino antisensemolecules has now been discovered, thus providing improved modes of genetherapy. The present invention employs cochleate delivery vehicles toprotect and deliver siRNA and morpholinos against target mRNA in cells,tissues, organs, and to organisms, e.g., animals and humans in a varietyof dosage forms (e.g., oral capsules and liquids) in a safe andeffective manner

Definitions

So that the invention may be more readily understood, certain terms arefirst defined.

The term “nucleoside” refers to a molecule having a purine or pyrimidinebase covalently linked to a ribose or deoxyribose sugar. Exemplarynucleosides include adenosine, guanosine, cytidine, uridine andthymidine. The term “nucleotide” refers to a nucleoside having one ormore phosphate groups joined in ester linkages to the sugar moiety.Exemplary nucleotides include nucleoside monophosphates, diphosphatesand triphosphates. The terms “polynucleotide” and “nucleic acidmolecule” are used interchangeably herein and refer to a polymer ofnucleotides joined together by a phosphodiester linkage between 5′ and3′ carbon atoms.

The term “nucleotide analog” or “altered nucleotide” or “modifiednucleotide” refers to a non-standard nucleotide, including non-naturallyoccurring ribonucleotides or deoxyribonucleotides. Preferred nucleotideanalogs are modified at any position so as to alter certain chemicalproperties of the nucleotide yet retain the ability of the nucleotideanalog to perform its intended function. Examples of positions of thenucleotide which may be derivatized include the 5 position, e.g.,5-(2-amino)propyl uridine, 5-bromo uridine, 5-propyne uridine,5-propenyl uridine, etc.; the 6 position, e.g., 6-(2amino)propyluridine; the 8-position for adenosine and/or guanosines, e.g., 8-bromoguanosine, 8-chloro guanosine, 8-fluoroguanosine, etc. Nucleotideanalogs also include deaza nucleotides, e.g., 7-deaza-adenosine; andN-modified (e.g., alkylated, e.g., N6methyl adenosine, or as otherwiseknown in the art) nucleotides; and other heterocyclically modifiednucleotide analogs such as those described in Herdewijn, AntisenseNucleic Acid Drug Dev., 2000 Aug. 10(4):297-310.

Nucleotide analogs may also comprise modifications to the sugar portionof the nucleotides. For example the 2′OH-group may be replaced by agroup selected from H, OR, R, F, Cl, Br, I, SH, SR, NH₂, NHR, NR₂, COOR,or OR, wherein R is substituted or unsubstituted C1-C6 alkyl, alkenyl,alkynyl, aryl, etc. Other possible modifications include those describedin U.S. Pat. Nos. 5,858,988, and 6,291,438.

The phosphate group of the nucleotide may also be modified, e.g., bysubstituting one or more of the oxygens of the phosphate group withsulfur (e.g., phosphorothioates), or by making other substitutions whichallow the nucleotide to perform its intended function such as describedin, for example, Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr.10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct. 11(5): 317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001 Apr.11.2):77-85, and U.S. Pat. No. 5,684,143. Certain of theabove-referenced modifications (e.g., phosphate group modifications)preferably decrease the rate of hydrolysis of, for example,polynucleotides comprising said analogs in vivo or in vivo.

The term “RNA” or “RNA molecule” or “ribonucleic acid molecule” refersto a polymer of ribonucleotides. The term “DNA” or “DNA molecule” ordeoxyribonucleic acid molecule” refers to a polymer ofdeoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., byDNA replication or transcription of DNA, respectively).

RNA can be post-transcriptionally modified. DNA and RNA can also bechemically synthesized. DNA and RNA can be single-stranded (i.e., ssRNAand ssDNA, respectively) or multi-stranded (e.g., double stranded, i.e.,dsRNA and dsDNA, respectively). “mRNA” or “messenger RNA” issingle-stranded RNA that specifies the amino acid sequence of one ormore polypeptide chains. This information is translated during proteinsynthesis when ribosomes bind to the mRNA.

As used herein, the term “small interfering RNA” (“siRNA”) (alsoreferred to in the art as “short interfering RNAs”) refers to a doublestranded RNA (or RNA analog) comprising between about 10-50 nucleotides(or nucleotide analogs) which is capable of directing or mediating RNAinterference.

The term “oligonucleotide” refers to a short polymer of nucleotidesand/or nucleotide analogs. The term “RNA analog” refers to anpolynucleotide (e.g., a chemically synthesized polynucleotide) having atleast one altered or modified nucleotide as compared to a correspondingunaltered or unmodified RNA, but retaining the same or similar nature orfunction as the corresponding unaltered or unmodified RNA. As discussedabove, the oligonucleotides may be linked with linkages which result ina lower rate of hydrolysis of the RNA analog as compared to an RNAmolecule with phosphodiester linkages. For example, the nucleotides ofthe analog may comprise methylenediol, ethylene diol, oxymethylthio,oxyethylthio, oxycarbonyloxy, phosphorodiamidate, phosphoroamidate,and/or phosphorothioate linkages. Preferred RNA analogues include sugar-and/or backbone-modified ribonucleotides and/or deoxyribonucleotides.Such alterations or modifications can further include addition ofnon-nucleotide material, such as to the end(s) of the RNA or internally(at one or more nucleotides of the RNA). An RNA analog need only besufficiently similar to natural RNA that it has the ability to mediate(mediates) RNA interference.

As used herein, an “identical” oligonucleotide has the same sequence asthe reference nucleotide subsequence to which the oligonucleotide isbeing compared. An “exactly complementary” oligonucleotide refers to anoligonucleotide whose complement has the same sequence as the referencenucleotide subsequence to which the oligonucleotide is being compared. A“substantially complementary” and a “substantially identical”oligonucleotide have the ability to specifically hybridize to areference gene, DNA, cDNA, or mRNA, and its exact complement.

As used herein, the term “RNA interference” (“RNAi”) refers to aselective intracellular degradation of RNA to mediate, reduce or silencethe expression of a target gene.

An siRNA “that mediates RNAi against a target mRNA” refers to an siRNAincluding a sequence sufficiently complementary to a target RNA (e.g.mRNA or RNA that can be spliced to produce one or more mRNAs) to triggerthe destruction of the target mRNA by the RNAi machinery or process.

A morpholino “that mediates translation of a target mRNA” refers to amorpholino including a sequence sufficiently complementary to a targetRNA (e.g. mRNA or RNA that can be spliced to produce one or more mRNAs)to interfere with translation of the mRNA into a protein.

As used herein, the term “isolated RNA” or “isolated siRNA” refers toRNA or siRNA molecules, respectively, which are substantially free ofother cellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized.

As used herein, the terms “cochleate,” “lipid precipitate” and“precipitate” are used interchangeably to refer to a lipid precipitatecomponent that generally includes alternating cationic and lipid bilayersheets with little or no internal aqueous space, typically stackedand/or rolled up, wherein the cationic sheet is comprised of one or moremultivalent cations. Additionally, the term “encochleated” meansassociated with the cochleate structure, e.g. by incorporation into thecationic sheet, and/or inclusion in the lipid bilayer.

As used herein, the term “multivalent cation” refers to a divalentcation or higher valency cation, or any compound that has at least twopositive charges, including mineral cations such as calcium, barium,zinc, iron and magnesium and other elements, such as drugs and othercompounds, capable of forming ions or other structures having multiplepositive charges capable of chelating and bridging negatively chargedlipids. Additionally or alternatively, the multivalent cation caninclude other multivalent cationic compounds, e.g., cationic orprotonized cargo moieties.

The lipid employed in the present invention preferably includes one ormore negatively charged lipids. As used herein, the term “negativelycharged lipid” includes lipids having a head group bearing a formalnegative charge in aqueous solution at an acidic, basic or physiologicalpH, and also includes lipids having a zwitterionic head group.

The term “in vitro” has its art recognized meaning, e.g., involvingpurified reagents or extracts, e.g., cell extracts. The term “in vivo”also has its art recognized meaning, e.g., involving living cells, e.g.,immortalized cells, primary cells, cell lines, and/or cells in anorganism.

A gene or mRNA “involved” in or “associated with” a disorder includes agene or mRNA, the normal or aberrant expression or function of whicheffects or causes a disease or disorder or at least one symptom of saiddisease or disorder.

The phrase “examining the function of a target mRNA” refers to examiningor studying the expression, activity, function or phenotype arisingtherefrom, in the host cell, tissue or organism.

Various methodologies of the instant invention include a step thatinvolves comparing a value, level, feature, characteristic, property,etc. to a “suitable control,” referred to interchangeably herein as an“appropriate control.” A “suitable control” or “appropriate control” isany control or standard familiar to one of ordinary skill in the artuseful for comparison purposes. In one embodiment, a “suitable control”or “appropriate control” is a value, level, feature, characteristic,property, etc. determined prior to performing an RNAi methodology, asdescribed herein. For example, a transcription rate, mRNA level,translation rate, protein level, biological activity, cellularcharacteristic or property, genotype, phenotype, etc. can be determinedprior to introducing an siRNA of the invention into a cell or organism.In another embodiment, a “suitable control” or “appropriate control” isa value, level, feature, characteristic, property, etc. determined in acell or organism, e.g. a control or normal cell or organism, exhibiting,for example, normal traits. In yet another embodiment, a “suitablecontrol” or “appropriate control” is a predefined value, level, feature,characteristic, property, etc.

The cochleates of the present invention can also include one or moreaggregation inhibitors. The term “aggregation inhibitor,” as usedherein, refers to an agent that inhibits aggregation of cochleates. Theaggregation inhibitor typically is present at least on the surface ofthe cochleate, and may only be present on the surface of the cochleate(e.g., when the aggregation inhibitor is introduced after cochleateformation). Aggregation inhibitors can be added before, after, and/orduring cochleate formation.

The terms “coat,” “coated,” “coating,” and the like, unless otherwiseindicated, refer to an agent (e.g. an aggregation inhibitor) present atleast on the outer surfaces of a cochleate. Such agents may beassociated with the bilayer by incorporation of at least part of theagent into the bilayer, and/or may be otherwise associated, e.g., byionic attraction to the cation or hydrophobic or ionic attraction to thelipid.

“Treatment”, or “treating” as used herein, refers to the application oradministration of a therapeutic agent (e.g., an siRNA cochleate) to apatient, or application or administration of a therapeutic agent to anisolated tissue or cell line from a patient, who has a disease ordisorder, a symptom of disease or disorder or a predisposition toward adisease or disorder, that effects or otherwise contributes to curing,healing, alleviating, relieving, altering, remedying, ameliorating,improving or affecting the disease or disorder, the symptoms of thedisease or disorder, or the predisposition toward disease.

The term “biologically effective amount” is that amount necessary orsufficient to produce a desired biological response.

An “antisense” oligonucleotide is an oligonucleotide that issubstantially complementary to a target nucleotide sequence and has theability to specifically hybridize to the target nucleotide sequence.

“Morpholino oligonucleotides” and “morpholinos” are usedinterchangeably, and refer to oligonucleotides having a morpholinobackbone.

Various aspects of the invention are described in further detail in thefollowing subsections.

siRNA-Cochleate Compositions

In one aspect, the present invention features encochleated siRNAcompositions. The siRNA-cochleate compositions generally include acochleate, and an siRNA associated with the cochleate.

Preferably, the siRNA molecule has a length from about 10-50 or morenucleotides. More preferably, the siRNA molecule has a length from about15-45 nucleotides. Even more preferably, the siRNA molecule has a lengthfrom about 19-40 nucleotides. Even more preferably, the siRNA moleculehas a length of from about 21-23 nucleotides.

The siRNA of the invention preferably mediates RNAi against a targetmRNA. The siRNA molecule can be designed such that every residue iscomplementary to a residue in the target molecule. Alternatively, one ormore substitutions can be made within the molecule to increase stabilityand/or enhance processing activity of said molecule. Substitutions canbe made within the strand or can be made to residues at the ends of thestrand.

The target mRNA cleavage reaction guided by siRNAs is sequence specific.In general, siRNA containing a nucleotide sequence identical to aportion of the target gene are preferred for inhibition. However, 100%sequence identity between the siRNA and the target gene is not requiredto practice the present invention. Sequence variations can be toleratedincluding those that might be expected due to genetic mutation, strainpolymorphism, or evolutionary divergence. For example, siRNA sequenceswith insertions, deletions, and single point mutations relative to thetarget sequence have also been found to be effective for inhibition.Alternatively, siRNA sequences with nucleotide analog substitutions orinsertions can be effective for inhibition.

Moreover, not all positions of an siRNA contribute equally to targetrecognition. Mismatches in the center of the siRNA are most critical andessentially abolish target RNA cleavage. In contrast, the 3′ nucleotidesof the siRNA do not contribute significantly to specificity of thetarget recognition. Generally, residues at the 3′ end of the siRNAsequence which is complementary to the target RNA (e.g., the guidesequence) are not critical for target RNA cleavage.

Sequence identity may readily be determined by sequence comparison andalignment algorithms known in the art. To determine the percent identityof two nucleic acid sequences (or of two amino acid sequences), thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in the first sequence or second sequence for optimalalignment). The nucleotides (or amino acid residues) at correspondingnucleotide (or amino acid) positions are then compared. When a positionin the first sequence is occupied by the same residue as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % homology=# of identical positions/total # ofpositions×100), optionally penalizing the score for the number of gapsintroduced and/or length of gaps introduced.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In one embodiment, the alignment generated over a certainportion of the sequence aligned having sufficient identity but not overportions having low degree of identity (i.e., a local alignment). Apreferred, non-limiting example of a local alignment algorithm utilizedfor the comparison of sequences is the algorithm of Karlin and Altschul(1990) Proc. NatL Acad Sci. USA 87:2264-68, modified as in Karlin andAltschul (1993) Proc. NatL Acad. Sci. USA 90:5873. Such an algorithm isincorporated into the BLAST programs (version 2.0) of Altschul, et al.(I 990) J Mol Biol. 215:403-10.

In another embodiment, the alignment is optimized by introducingappropriate gaps and percent identity is determined over the length ofthe aligned sequences (i.e., a gapped alignment). To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389. Inanother embodiment, the alignment is optimized by introducingappropriate gaps and percent identity is determined over the entirelength of the sequences aligned (i.e., a global alignment). A preferred,non-limiting example of a mathematical algorithm utilized for the globalcomparison of sequences is the algorithm of Myers and Miller, CABIOS(1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

Greater than 90% sequence identity, e.g., 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or even 100% sequence identity, between the siRNA and theportion of the target mRNA is preferred. Alternatively, the siRNA may bedefined functionally as a nucleotide sequence (or oligonucleotidesequence) that is capable of hybridizing with a portion of the targetmRNA transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C. or 70° C. hybridization for 12-16 hours; followed by washing).Additional hybridization conditions include hybridization at 70° C. in1×SSC or 50° C. in 1×SSC, 50% formamide followed by washing at 70° C. in0.3×SSC or hybridization at 70° C. in 4×SSC or 50° C. in 4×SSC, 50%formamide followed by washing at 67° C. in 1×SSC. The hybridizationtemperature for hybrids anticipated to be less than 50 base pairs inlength should be 5-10° C. less than the melting temperature (Tm) of thehybrid, where Tm is determined according to the following equations. Forhybrids less than 18 base pairs in length, Tm(° C.)=2(# of A+Tbases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs inlength, Tm(° C.)=81.5+16.6(log₁₀[Na+])+0.41 (% G+C)−(600/N), where N isthe number of bases in the hybrid, and [Na+] is the concentration ofsodium ions in the hybridization buffer ([Na+] for 1×SSC=0.165 M).Additional examples of stringency conditions for polynucleotidehybridization are provided in Sambrook, J., E. F. Fritsch, and T.Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold 15 Spring Harbor, N.Y., chapters 9 and 11,and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al.,eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporatedherein by reference. The length of the identical nucleotide sequencesmay be at least about or about equal to 10, 12, 15, 17, 20, 22, 25, 27,30, 32, 35, 37, 40, 42, 45, 47 or 50 bases.

In one embodiment, the siRNA molecules of the present invention aremodified to improve stability in serum or in growth medium for cellcultures. In order to enhance the stability, the 3′-residues may bestabilized against degradation, e.g., they may be selected such thatthey consist of purine nucleotides, particularly adenosine or guanosinenucleotides. Alternatively, substitution of pyrimidine nucleotides bymodified analogues, e.g., substitution of uridine by 2′-deoxythymidineis tolerated and does not affect the efficiency of RNA interference. Forexample, the absence of a 2′hydroxyl may significantly enhance thenuclease resistance of the siRNAs in tissue culture medium.

In another embodiment of the present invention the siRNA molecule maycontain at least one modified nucleotide analogue. The nucleotideanalogues may be located at positions where the target-specificactivity, e.g., the RNAi mediating activity is not substantiallyeffected, e.g., in a region at the 5′-end and/or the 3′-end of the RNAmolecule. Particularly, the ends may be stabilized by incorporatingmodified nucleotide analogues.

Nucleotide analogues include sugar- and/or backbone-modifiedribonucleotides (i.e., include modifications to the phosphate-sugarbackbone). For example, the phosphodiester linkages of natural RNA maybe modified to include at least one of a nitrogen or sulfur heteroatom.In preferred backbone-modified ribonucleotides the phosphoester groupconnecting to adjacent ribonucleotides is replaced by a modified group,e.g., of phosphothioate group. In preferred sugar modifiedribonucleotides, the 2′OH-group is replaced by a group selected from H,OR, R, halo, SH, SR, NH₂, NHR, NR₂ or NO₂, wherein R is C1-C6 alkyl,alkenyl or alkynyl and halo is F, Cl, Br or I.

Nucleotide analogues also include nucleobase-modified ribonucleotides,i.e., ribonucleotides, containing at least one non-naturally occurringnucleobase instead of a naturally occurring nucleobase. Bases may bemodified to block the activity of adenosine deaminase. Exemplarymodified nucleobases include, but are not limited to, uridine and/orcytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine,5-bromo uridine; adenosine and/or guanosines modified at the 8 position,e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O-and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. Itshould be noted that the above modifications may be combined.

RNA may be produced enzymatically or by partial/total organic synthesis,any modified ribonucleotide can be introduced by in vitro enzymatic ororganic synthesis. In one embodiment, an siRNA is prepared chemically.Methods of synthesizing RNA molecules are known in the art, inparticular, the chemical synthesis methods as described in Verina andEckstein (1998), Annul Rev. Biochem. 67:99. In another embodiment, ansiRNA is prepared enzymatically. For example, an siRNA can be preparedby enzymatic processing of a long, double-stranded RNA having sufficientcomplementarity to the desired target mRNA. Processing of long RNA canbe accomplished in vitro, for example, using appropriate cellularlysates and siRNAs can be subsequently purified by gel electrophoresisor gel filtration. siRNA can then be denatured according toart-recognized methodologies. In an exemplary embodiment, siRNA can bepurified from a mixture by extraction with a solvent or resin,precipitation, electrophoresis, chromatography, or a combinationthereof. Alternatively, the siRNA may be used with no or a minimum ofpurification to avoid losses due to sample processing.

Alternatively, the siRNAs can also be prepared by enzymatictranscription from synthetic DNA templates or from DNA plasmids isolatedfrom recombinant bacteria. Typically, phage RNA polymerases are usedsuch as T7, T3 or SP6 RNA polyimerase (Milligan and Uhlenbeck (1989)Methods EnzynioL 180:51-62). The RNA may be dried for storage ordissolved in an aqueous solution. The solution may contain buffers orsalts to inhibit annealing, and/or promote stabilization of the doublestrands.

Commercially available design tools and kits, such as those availablefrom Ambion, Inc. (Austin, Tex.), and the Whitehead Institute ofBiomedical Research at MIT (Cambridge, Mass.) allow for the design andproduction of siRNA. By way of example, a desired mRNA sequence can beentered into a sequence program that will generate sense and antisensetarget strand sequences. These sequences can then be entered into aprogram that determines the sense and antisense siRNA oligonucleotidetemplates. The programs can also be used to add, e.g., hairpin insertsor T1 promoter primer sequences. Kits also can then be employed to buildsiRNA expression cassettes.

In various embodiments, siRNAs are synthesized in vivo, in situ, and invitro. Endogenous RNA polymerase of the cell may mediate transcriptionin vivo or in situ, or cloned RNA polymerase can be used fortranscription in vivo or in vitro. For transcription from a transgene invivo or an expression construct, a regulatory region (e.g., promoter,enhancer, silencer, splice donor and acceptor, polyadenylation) may beused to transcribe the siRNAs. Inhibition may be targeted by specifictranscription in an organ, tissue, or cell type; stimulation of anenvironmental condition (e.g., infection, stress, temperature, chemicalinducers); and/or engineering transcription at a developmental stage orage. A transgenic organism that expresses siRNAs from a recombinantconstruct may be produced by introducing the construct into a zygote, anembryonic stem cell, or another multipotent cell derived from theappropriate organism.

In one embodiment, the target mRNA of the invention specifies the aminoacid sequence of at least one protein such as a cellular protein (e.g.,a nuclear, cytoplasmic, transmembrane, or membrane-associated protein).In another embodiment, the target mRNA of the invention specifies theamino acid sequence of an extracellular protein (e.g., an extracellularmatrix protein or secreted protein). As used herein, the phrase“specifies the amino acid sequence” of a protein means that the mRNAsequence is translated into the amino acid sequence according to therules of the genetic code. The following classes of proteins are listedfor illustrative purposes: developmental proteins (e.g., adhesionmolecules, cyclin kinase inhibitors, Wnt family members, Pax familymembers, Winged helix family members, Hox family members,cytokines/lymphokines and their receptors, growth/differentiationfactors and their receptors, neurotransmitters and their receptors);oncogene-encoded proteins (e.g., ABLI, BCLI, BCL2, BCL6, CBFA2. CBL,CSFIR, ERBA, ERBB, EBRB2, ERBB2, ERBB3, ETSI, ETSI, ETV6, FGR, FOS, FYN,HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS,PIM 1, PML, RET, SRC, TALI, TCL3, and YES); tumor suppressor proteins(e.g., APC, BRCA1, BRCA2, MADH4, MCC, NF 1, NF2, RB 1, TP53, and WTI);and enzymes (e.g., ACC synthases and oxidases, ACP desaturases andhydroxylases, ADPglucose pyrophorylases, acetylases and deacetylases,ATPases, alcohol dehydrogenases, amylases, amyloglucosidases, catalases,cellulases, chalcone synthases, chitinases, cyclooxygenases,decarboxylases, dextrinases, DNA and RNA polymerases, galactosidases,glucanases, glucose oxidases, granule-bound starch synthases, GTPases,helicases, hemicellulases, integrases, inulinases, invertases,isomerases, kinases, lactases, lipases, lipoxygenases, lysozymes,nopaline synthases, octopine synthases, pectinesterases, peroxidases,phosphatases, phospholipases, phosphorylases, phytases, plant growthregulator synthases, polygalacturonases, proteinases and peptidases,pullanases, recombinases, reverse transcriptases, RUBISCOs,topoisomerases, and xylanases), proteins involved in tumor growth(including vascularization) or in metastatic activity or potential,including cell surface receptors and ligands as well as secretedproteins, cell cycle regulatory, gene regulatory, and apoptosisregulatory proteins, immune response, inflammation, complement, orclotting regulatory proteins.

As used herein, the term “oncogene” refers to a gene which stimulatescell growth and, when its level of expression in the cell is reduced,the rate of cell growth is reduced or the cell becomes quiescent. In thecontext of the present invention, oncogenes include intracellularproteins, as well as extracellular growth factors which may stimulatecell proliferation through autocrine or paracrine function. Examples ofhuman oncogenes against which siRNA and morpholino constructs candesigned include c-myc, c-myb, mdm2, PKA-I (protein kinase A type I),Abl-1, Bcl2, Ras, c-Raf kinase, CDC25 phosphatases, cyclins, cyclindependent kinases (cdks), telomerase, PDGF/sis, erb-B, fos, jun, mos,and src, to name but a few. In the context of the present invention,oncogenes also include a fusion gene resulted from chromosomaltranslocation, for example, the Bcr/Abl fusion oncogene.

Further proteins include cyclin dependent kinases, c-myb, c-myc,proliferating cell nuclear antigen (PCNA), transforming growthfactor-beta (TGF-beta), and transcription factors nuclear factor kappaB(NF-.kappa.B), E2F, HER-2/neu, PKA, TGF-alpha, EGFR, TGF-beta, IGFIR,P12, MDM2, BRCA, Bcl-2, VEGF, MDR, ferritin, transferrin receptor, IRE,C-fos, HSP27, C-raf and metallothionein genes.

The siRNA employed in the present invention can be directed against thesynthesis of one or more proteins. Additionally or alternatively, therecan be more than one siRNA directed against a protein, e.g., duplicatesiRNA or siRNA that correspond to overlapping or non-overlapping targetsequences against the same target protein. Accordingly, in oneembodiment two, three, four or any plurality of siRNAs against the sametarget mRNA can be including in the cochleate compositions of theinvention. Additionally, several siRNAs directed against severalproteins can be employed. Alternatively, the siRNA can be directedagainst structural or regulatory RNA molecules that do not code forproteins.

In a preferred aspect of the invention, the target mRNA molecule of theinvention specifies the amino acid sequence of a protein associated witha pathological condition. For example, the protein may be apathogen-associated protein (e.g., a viral protein involved inimmunosuppression or immunoavoidance of the host, replication of thepathogen, transmission of the pathogen, or maintenance of theinfection), or a host protein which facilitates entry of the pathogeninto the host, drug metabolism by the pathogen or host, replication orintegration of the pathogen's genome, establishment or spread ofinfection in the host, or assembly of the next generation of pathogen.Alternatively, the protein may be a tumor-associated protein or anautoimmune disease-associated protein.

In one embodiment, the target mRNA molecule of the invention specifiesthe amino acid sequence of an endogenous protein (i.e. a protein presentin the genome of a cell or organism). In another embodiment, the targetmRNA molecule of the invention specifies the amino acid sequence of aheterologous protein expressed in a recombinant cell or a geneticallyaltered organism. In another embodiment, the target mRNA molecule of theinvention specifies the amino acid sequence of a protein encoded by atransgene (i.e., a gene construct inserted at an ectopic site in thegenome of the cell). In yet another embodiment, the target mRNA moleculeof the invention specifies the amino acid sequence of a protein encodedby a pathogen genome which is capable of infecting a cell or an organismfrom which the cell is derived.

By inhibiting the expression of such proteins, valuable informationregarding the function of said proteins and therapeutic benefits whichmay be obtained from said inhibition may be obtained.

Accordingly, in one embodiment, the siRNA-cochleate compositions of thepresent invention can be utilized in studies of mammalian cells toclarify the role of specific structural and catalytic proteins. Inanother embodiment, they can be used in a therapeutic application tospecifically target pathogenic organisms, including fungi, bacteria, andviruses.

Cochleate delivery vehicles are stable lipid-cation precipitates thatcan be composed of simple, naturally occurring materials, e.g.,phosphatidylserine, and calcium. Mixtures of naturally occurringmolecules (e.g., soy lipids) and/or synthetic or modified lipids can beutilized.

The cochleate structure provides protection from degradation forassociated “encochleated” moieties. Divalent cation concentrations invivo in serum and mucosal secretions are such that the cochleatestructure is maintained. Hence, the majority of cochleate-associatedmolecules, e.g., cargo moieties, are present in the inner layers of aprimarily solid, non-aqueous, stable, impermeable structure. Since thecochleate structure includes a series of solid layers, components withinthe interior of the cochleate structure remain substantially intact,even though the outer layers of the cochleate may be exposed to harshenvironmental conditions or enzymes.

The cochleate interior is primarily free of water and resistant topenetration by oxygen. Oxygen and water are primarily responsible forthe decomposition and degradation of molecules which can lead to reducedshelf-life. Accordingly, encochleation should also impart extensiveshelf-life stability to encochleated siRNAs.

With respect to storage, cochleates can be stored in cation-containingbuffer, or lyophilized or otherwise converted to a powder, and stored atroom temperature. If desired, the cochleates also can be reconstitutedwith liquid prior to administration. Cochleate preparations have beenshown to be stable for more than two years at 4° C. in acation-containing buffer, and at least one year as a lyophilized powderat room temperature.

In one embodiment, the cochleate comprises a negatively charged lipidcomponent and a multivalent cation component.

In one embodiment, the lipid is a mixture of lipids, comprising at least75% negatively charged lipid. In another embodiment, the lipid includesat least 85% negatively charged lipid. In other embodiments, the lipidincludes at least 90%, 95% or even 99% negatively charged lipid. Allranges and values between 60% and 100% negatively charged lipid aremeant to be encompassed herein.

The negatively charged lipid can include soy-based lipids. Preferably,the lipid includes phospholipids, such as soy phospholipids (soy-basedphospholipids). The negatively charged lipid can include phosphotidylserine (PS), dioleoylphosphatidylserine (DOPS), phosphatidic acid (PA),phosphatidylinositol (PI), and/or phosphatidyl glycerol (PG) and or amixture of one or more of these lipids with other lipids. Additionallyor alternatively, the lipid can include phosphatidylcholine (PC),phosphatidylethanolamine (PE), diphosphotidylglycerol (DPG), dioleoylphosphatidic acid (DOPA), distearoyl phosphatidylserine (DSPS),dimyristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidylgycerol(DPPG) and the like.

The lipids can be natural or synthetic. For example, the lipid caninclude esterified fatty acid acyl chains, or organic chains attached bynon-ester linkages such as ether linkages (as described in U.S. Pat. No.5,956,159), disulfide linkages, and their analogs.

In one embodiment the lipid chains are from about 6 to about 26 carbonatoms, and the lipid chains can be saturated or unsaturated. Fatty acyllipid chains useful in the present invention include, but are notlimited to, n-tetradecanoic, n-hexadecanoic acid, n-octadecanoic acid,n-eicosanoic acid, n-docosanoic acid, n-tetracosanoic acid,n-hexacosanoic acid, cis-9-hexadecenoic acid, cis-9-octadecenoic acid,cis,cis-9,12-octadecedienoic acid, all-cis-9,12,15-octadecetrienoicacid, all-cis-5,8,11,14-eicosatetraenoic acid,all-cis-4,7,10,13,16,19-docosahexaenoic acid, 2,4,6,8-tetramethyldecanoic acid, and lactobacillic acid, and the like.

In some embodiments, pegylated lipid also is included. Pegylated lipidincludes lipids covalently linked to polymers of polyethylene glycol(PEG). PEG's are conventionally classified by their molecular weight,thus PEG 6,000 MW, e.g., has a molecular weight of about 6000. Addingpegylated lipid generally will result in an increase of the amount ofcompound (e.g., peptide, nucleotide, and nutrient) that can beincorporated into the precipitate. An exemplary pegylated lipid isdipalmitoylphosphatidylehtanolamine (DPPE) bearing PEG 5,000 MW.

The siRNA-cochleate compositions of the present invention can beprovided in a variety of forms (e.g. powder, liquid, suspension) with orwithout additional components. Suitable forms and additives, excipients,carriers and the like are described herein.

Morpholino-Cochleate Compositions

The present invention also features encochleated morpholino antisenseoligonucleotides (morpholinos) and methods (e.g., research and/ortherapeutic methods) for using said morpholino-cochleates. In oneaspect, the present invention provides a morpholino-cochleatecomposition that generally includes a cochleate, and a morpholinoassociated with the cochleate.

Morpholinos function by an RNase H-independent mechanism and are solublein aqueous solutions, with most being freely soluble at mMconcentrations (typically 10 mg/ml to over 100 mg/ml). Morpholinos havehigh affinity for RNA and efficiently invade even quite stable secondarystructures in mRNAs, which results in effective and predictabletargeting essentially anywhere from the 5′cap to about +25 of theprotein coding region of mRNAs. Morpholinos are free of significantnon-antisense effects while the alternative phosphorothioates areplagued by a host of well-documented non-antisense effects. Morpholinosinclude a morpholine backbone, which is not recognized by nucleases andtherefore is stable in the cell compared to phosphorothioates, whichtypically are degraded in biological systems in a matter of hours.Consequently, considerably fewer morpholinos are required (approximately100× less) to achieve similar antisense effects. Morpholinos also aresuperior to phosphorothioates because targeting is more predictable, theactivity in cells is more reliable, and the sequence specificity issuperior. Summerton, Biochimica et Biophysica Acta 1489: 141-158 (1999).Morpholinos can be designed and prepared according to known methods.E.g., Summerton and Weller, Antisense and Nucleic Acid Drug Development7187-195 (1997).

Morpholino oligonucleotides suitable for use in the present inventioninclude antisense morpholino oligonucleotides. The morpholino can bebetween about 7 and 100 nucleotides long, between 10 and 50, between 20and 35, and between 15 and 30 nucleotides long. In a preferredembodiment, the morpholino oligonucleotide is between about 18 and about25 nucleotides long. The oligonucleotides can be 17, 18, 19, 20, 21, 22,23, 24 or 25 nucleotides long.

The morpholinos of the invention preferably mediate RNA interferenceagainst a target gene. That is, preferably, the morpholino has asequence sufficiently complementary to a target RNA (e.g. mRNA or RNAthat can be spliced to produce one or more mRNAs) associated with atarget gene to trigger the destruction of the target mRNA by the RNAimachinery or process. The morpholino molecule can be designed such thatevery residue is complementary to a residue in the target molecule.Alternatively, one or more substitutions can be made within the moleculeto increase stability and/or enhance processing activity of saidmolecule. Substitutions can be made within the strand or can be made toresidues at the ends of the strand.

The target mRNA cleavage reaction guided by morpholinos is sequencespecific. In general, morpholinos containing a nucleotide sequenceidentical to a portion of the target gene are preferred for inhibition.However, 100% sequence identity between the morpholino and the targetgene is not required to practice the present invention. Sequencevariations can be tolerated including those that might be expected dueto genetic mutation, strain polymorphism, or evolutionary divergence.For example, morpholino sequences with insertions, deletions, and singlepoint mutations relative to the target sequence have also been found tobe effective for inhibition. Alternatively, morpholino sequences withnucleotide analog substitutions or insertions can be effective forinhibition.

Sequence identity may readily be determined by sequence comparison andalignment algorithms known in the art. To determine the percent identityof two nucleic acid sequences (or of two amino acid sequences), thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in the first sequence or second sequence for optimalalignment). The nucleotides (or amino acid residues) at correspondingnucleotide (or amino acid) positions are then compared. When a positionin the first sequence is occupied by the same residue as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % homology=# of identical positions/total # ofpositions×100), optionally penalizing the score for the number of gapsintroduced and/or length of gaps introduced.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In one embodiment, the alignment generated over a certainportion of the sequence aligned having sufficient identity but not overportions having low degree of identity (i.e., a local alignment). Apreferred, non-limiting example of a local alignment algorithm utilizedfor the comparison of sequences is the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873. Such an algorithm isincorporated into the BLAST programs (version 2.0) of Altschul, et al.(1990) J Mol. Biol. 215:403-10.

In another embodiment, the alignment is optimized by introducingappropriate gaps and percent identity is determined over the length ofthe aligned sequences (i.e., a gapped alignment). To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389. Inanother embodiment, the alignment is optimized by introducingappropriate gaps and percent identity is determined over the entirelength of the sequences aligned (i.e., a global alignment). A preferred,non-limiting example of a mathematical algorithm utilized for the globalcomparison of sequences is the algorithm of Myers and Miller, CABIOS(1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

Greater than 90% sequence identity, e.g., 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or even 100% sequence identity, between the morpholino andthe portion of the target RNA is preferred. Alternatively, themorpholino may be defined functionally as a nucleotide sequence (oroligonucleotide sequence) that is capable of hybridizing with a portionof the target mRNA transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1mM EDTA, 50° C. or 70° C. hybridization for 12-16 hours; followed bywashing). Additional hybridization conditions include hybridization at70° C. in 1×SSC or 50° C. in 1×SSC, 50% formamide followed by washing at70° C. in 0.3×SSC or hybridization at 70° C. in 4×SSC or 50° C. in4×SSC, 50% formamide followed by washing at 67° C. in 1×SSC. Thehybridization temperature for hybrids anticipated to be less than 50base pairs in length should be 5-10° C. less than the meltingtemperature (Tm) of the hybrid, where Tm is determined according to thefollowing equations. For hybrids less than 18 base pairs in length, Tm(°C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49base pairs in length, Tm(° C.)=81.5+16.6(log₁₀[Na+])+0.41 (%G+C)−(600/N), where N is the number of bases in the hybrid, and [Na+] isthe concentration of sodium ions in the hybridization buffer ([Na+] for1×SSC=0.165 M). Additional examples of stringency conditions forpolynucleotide hybridization are provided in Sambrook, J., E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold 15 Spring Harbor, N.Y.,chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F.M. Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and6.3-6.4, incorporated herein by reference. The length of the identicalnucleotide sequences may be at least about or about equal to 10, 12, 15,17, 20, 22, 25, 27, 30, 32, 35, 37, 40, 42, 45, 47 or 50 bases.

In one embodiment, the morpholino molecules of the present invention aremodified to improve stability in serum or in growth medium for cellcultures. In order to enhance the stability, the 3′-residues may bestabilized against degradation, e.g., they may be selected such thatthey consist of purine nucleotides, particularly adenosine or guanosinenucleotides. Alternatively, substitution of pyrimidine nucleotides bymodified analogues, e.g., substitution of uridine by 2′-deoxythymidineis tolerated and does not affect the efficiency of RNA interference. Forexample, the absence of a 2′hydroxyl may significantly enhance thenuclease resistance of the morpholinos in tissue culture medium.

In another embodiment of the present invention the morpholino moleculemay contain at least one modified nucleotide analogue. The nucleotideanalogues may be located at positions where the target-specificactivity, e.g., the RNAi mediating activity is not substantiallyeffected, e.g., in a region at the 5′-end and/or the 3′-end of the RNAmolecule. Particularly, the ends may be stabilized by incorporatingmodified nucleotide analogues.

Nucleotide analogues include sugar- and/or backbone-modifiedribonucleotides (i.e., include modifications to the phosphate-sugarbackbone). For example, the phosphodiester linkages of natural RNA maybe modified to include at least one of a nitrogen or sulfur heteroatom.In preferred backbone-modified ribonucleotides the phosphoester groupconnecting to adjacent ribonucleotides is replaced by a modified group,e.g., of phosphothioate group. In preferred sugar modifiedribonucleotides, the 2′OH-group is replaced by a group selected from H,OR, R, halo, SH, SR, NH₂, NHR, NR₂ or NO₂, wherein R is C1-C6 alkyl,alkenyl or alkynyl and halo is F, Cl, Br or I.

Nucleotide analogues also include nucleobase-modified ribonucleotides,i.e., ribonucleotides, containing at least one non-naturally occurringnucleobase instead of a naturally occurring nucleobase. Bases may bemodified to block the activity of adenosine deaminase. Exemplarymodified nucleobases include, but are not limited to, uridine and/orcytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine,5-bromo uridine; adenosine and/or guanosines modified at the 8 position,e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O-and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. Itshould be noted that the above modifications may be combined.

RNA may be produced enzymatically or by partial/total organic synthesis,any modified ribonucleotide can be introduced by in vitro enzymatic ororganic synthesis. In one embodiment, a morpholino is preparedchemically. Methods of synthesizing RNA molecules are known in the art,in particular, the chemical synthesis methods as described in Verina andEckstein (1998), Annul Rev. Biochem. 67:99. In another embodiment, amorpholino is prepared enzymatically. For example, a morpholino can beprepared by enzymatic processing of a long, double-stranded RNA havingsufficient complementarity to the desired target mRNA. Processing oflong RNA can be accomplished in vitro, for example, using appropriatecellular lysates and morpholinos can be subsequently purified by gelelectrophoresis or gel filtration. Morpholinos can then be denaturedaccording to art-recognized methodologies. In an exemplary embodiment,morpholinos can be purified from a mixture by extraction with a solventor resin, precipitation, electrophoresis, chromatography, or acombination thereof. Alternatively, the morpholino may be used with noor a minimum of purification to avoid losses due to sample processing.

In one embodiment, morpholinos are synthesized either in vivo, in situ,or in vitro. Endogenous RNA polymerase of the cell may mediatetranscription in vivo or in situ, or cloned RNA polymerase can be usedfor transcription in vivo or in vivo. For transcription from a transgenein vivo or an expression construct, a regulatory region (e.g., promoter,enhancer, silencer, splice donor and acceptor, polyadenylation) may beused to transcribe the morpholinos. Inhibition may be targeted byspecific transcription in an organ, tissue, or cell type; stimulation ofan environmental condition (e.g., infection, stress, temperature,chemical inducers); and/or engineering transcription at a developmentalstage or age. A transgenic organism that expresses morpholinos from arecombinant construct may be produced by introducing the construct intoa zygote, an embryonic stem cell, or another multipotent cell derivedfrom the appropriate organism.

In one embodiment, the target mRNA of the invention specifies the aminoacid sequence of at least one protein such as a cellular protein (e.g.,a nuclear, cytoplasmic, transmembrane, or membrane-associated protein).In another embodiment, the target mRNA of the invention specifies theamino acid sequence of an extracellular protein (e.g., an extracellularmatrix protein or secreted protein). As used herein, the phrase“specifies the amino acid sequence” of a protein means that the mRNAsequence is translated into the amino acid sequence according to therules of the genetic code. The following classes of proteins are listedfor illustrative purposes: developmental proteins (e.g., adhesionmolecules, cyclin kinase inhibitors, Wnt family members, Pax familymembers, Winged helix family members, Hox family members,cytokines/lymphokines and their receptors, growth/differentiationfactors and their receptors, neurotransmitters and their receptors);oncogene-encoded proteins (e.g., ABLI, BCLI, BCL2, BCL6, CBFA2. CBL,CSFIR, ERBA, ERBB, EBRB2, ETSI, ETSI, ETV6. FGR, FOS, FYN, HCR, HRAS,JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS, PIM 1, PML,RET, SRC, TALI, TCL3, and YES); tumor suppressor proteins (e.g., APC,BRCA1, BRCA2, MADH4, MCC, NF 1, NF2, RB 1, TP53, and WTI); and enzymes(e.g., ACC synthases and oxidases, ACP desaturases and hydroxylases,ADPglucose pyrophorylases, acetylases and deacetylases, ATPases, alcoholdehydrogenases, amylases, amyloglucosidases, catalases, cellulases,chalcone synthases, chitinases, cyclooxygenases, decarboxylases,dextrinases, DNA and RNA polymerases, galactosidases, glucanases,glucose oxidases, granule-bound starch synthases, GTPases, helicases,hemicellulases, integrases, inulinases, invertases, isomerases, kinases,lactases, lipases, lipoxygenases, lysozymes, nopaline synthases,octopine synthases, pectinesterases, peroxidases, phosphatases,phospholipases, phosphorylases, phytases, plant growth regulatorsynthases, polygalacturonases, proteinases and peptidases, pullanases,recombinases, reverse transcriptases, RUBISCOs, topoisomerases, andxylanases), proteins involved in tumor growth (includingvascularization) or in metastatic activity or potential, including cellsuface receptors and ligands as well as secreted proteins, cell cycleregulatory, gene regulatory, and apoptosis regulatory proteins, immuneresponse, inflammation, complement, or clotting regulatory proteins.

As used herein, the term “oncogene” refers to a gene which stimulatescell growth and, when its level of expression in the cell is reduced,the rate of cell growth is reduced or the cell becomes quiescent. In thecontext of the present invention, oncogenes include intracellularproteins, as well as extracellular growth factors which may stimulatecell proliferation through autocrine or paracrine function. Examples ofhuman oncogenes against which siRNA and morpholino constructs candesigned include c-myc, c-myb, mdm2, PKA-I (protein kinase A type I),Abl-1, Bcl2, Ras, c-Raf kinase, CDC25 phosphatases, cyclins, cyclindependent kinases (cdks), telomerase, PDGF/sis, erb-B, fos, jun, mos,and src, to name but a few. In the context of the present invention,oncogenes also include a fusion gene resulted from chromosomaltranslocation, for example, the Bcr/Abl fusion oncogene.

Further proteins include cyclin dependent kinases, c-myb, c-myc,proliferating cell nuclear antigen (PCNA), transforming growthfactor-beta (TGF-beta), and transcription factors nuclear factor kappaB(NF-.kappa.B), E2F, HER-2/neu, PKA, TGF-alpha, EGFR, TGF-beta, IGFIR,P12, MDM2, BRCA, Bcl-2, VEGF, MDR, ferritin, transferrin receptor, IRE,C-fos, HSP27, C-raf and metallothionein genes.

The morpholinos employed in the present invention can be directedagainst the synthesis of one or more proteins. Additionally oralternatively, there can be more than one morpholino directed against aprotein, e.g., duplicate morholinos or morpholinos that correspond tooverlapping or non-overlapping target sequences against the same targetprotein. Additionally, several morpholinos directed against severalproteins can be employed. Accordingly, in one embodiment two, three,four or any plurality of morpholinos against the same target mRNA can beincluding in the cochleate compositions of the invention. Alternatively,the morpholino can be directed against structural or regulatory RNAmolecules that do not code for proteins.

In a preferred aspect of the invention, the target mRNA molecule of theinvention specifies the amino acid sequence of a protein associated witha pathological condition. For example, the protein may be apathogen-associated protein (e.g., a viral protein involved inimmunosuppression or immunoavoidance of the host, replication of thepathogen, transmission of the pathogen, or maintenance of theinfection), or a host protein which facilitates entry of the pathogeninto the host, drug metabolism by the pathogen or host, replication orintegration of the pathogen's genome, establishment or spread ofinfection in the host, or assembly of the next generation of pathogen.Alternatively, the protein may be a tumor-associated protein or anautoimmune disease-associated protein.

In one embodiment, the target mRNA molecule of the invention specifiesthe amino acid sequence of an endogenous protein (i.e. a protein presentin the genome of a cell or organism). In another embodiment, the targetmRNA molecule of the invention specifies the amino acid sequence of aheterologous protein expressed in a recombinant cell or a geneticallyaltered organism. In another embodiment, the target mRNA molecule of theinvention specifies the amino acid sequence of a protein encoded by atransgene (i.e., a gene construct inserted at an ectopic site in thegenome of the cell). In yet another embodiment, the target mRNA moleculeof the invention specifies the amino acid sequence of a protein encodedby a pathogen genome which is capable of infecting a cell or an organismfrom which the cell is derived.

By inhibiting the expression of such proteins, valuable informationregarding the function of said proteins and therapeutic benefits whichmay be obtained from said inhibition may be obtained.

Accordingly, in one embodiment, the morpholino-cochleate compositions ofthe present invention can be utilized in studies of mammalian cells toclarify the role of specific structural and catalytic proteins. Inanother embodiment, they can be used in a therapeutic application tospecifically target pathogenic organisms, including fungi, bacteria, andviruses.

Cochleate delivery vehicles are stable lipid-cation precipitates thatcan be composed of simple, naturally occurring materials, e.g.,phosphatidylserine, and calcium. Mixtures of naturally occurringmolecules (e.g., soy lipids) and/or synthetic or modified lipids can beutilized.

The cochleate structure provides protection from degradation forassociated “encochleated” moieties. Divalent cation concentrations invivo in serum and mucosal secretions are such that the cochleatestructure is maintained. Hence, the majority of cochleate-associatedmolecules, e.g., cargo moieties, are present in the inner layers of aprimarily solid, non-aqueous, stable, impermeable structure. Since thecochleate structure includes a series of solid layers, components withinthe interior of the cochleate structure remain substantially intact,even though the outer layers of the cochleate may be exposed to harshenvironmental conditions or enzymes.

The cochleate interior is primarily free of water and resistant topenetration by oxygen. Oxygen and water are primarily responsible forthe decomposition and degradation of molecules which can lead to reducedshelf-life. Accordingly, encochleation should also impart extensiveshelf-life stability to encochleated morpholinos.

With respect to storage, cochleates can be stored in cation-containingbuffer, or lyophilized or otherwise converted to a powder, and stored atroom temperature. If desired, the cochleates also can be reconstitutedwith liquid prior to administration. Cochleate preparations have beenshown to be stable for more than two years at 4° C. in acation-containing buffer, and at least one year as a lyophilized powderat room temperature.

In one embodiment, the cochleate comprises a negatively charged lipidcomponent and a multivalent cation component.

In one embodiment, the lipid is a mixture of lipids, comprising at least75% negatively charged lipid. In another embodiment, the lipid includesat least 85% negatively charged lipid. In other embodiments, the lipidincludes at least 90%, 95% or even 99% negatively charged lipid. Allranges and values between 60% and 100% negatively charged lipid aremeant to be encompassed herein.

The negatively charged lipid can include soy-based lipids. Preferably,the lipid includes phospholipids, such as soy phospholipids (soy-basedphospholipids). The negatively charged lipid can include phosphotidylserine (PS), dioleoylphosphatidylserine (DOPS), phosphatidic acid (PA),phosphatidylinositol (PI), and/or phosphatidyl glycerol (PG) and or amixture of one or more of these lipids with other lipids. Additionallyor alternatively, the lipid can include phosphatidylcholine (PC),phosphatidylethanolamine (PE), diphosphotidylglycerol (DPG), dioleoylphosphatidic acid (DOPA), distearoyl phosphatidylserine (DSPS),dimyristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidylgycerol(DPPG) and the like.

The lipids can be natural or synthetic. For example, the lipid caninclude esterified fatty acid acyl chains, or organic chains attached bynon-ester linkages such as ether linkages (as described in U.S. Pat. No.5,956,159), disulfide linkages, and their analogs.

In one embodiment the lipid chains are from about 6 to about 26 carbonatoms, and the lipid chains can be saturated or unsaturated. Fatty acyllipid chains useful in the present invention include, but are notlimited to, n-tetradecanoic, n-hexadecanoic acid, n-octadecanoic acid,n-eicosanoic acid, n-docosanoic acid, n-tetracosanoic acid,n-hexacosanoic acid, cis-9-hexadecenoic acid, cis-9-octadecenoic acid,cis,cis-9,12-octadecedienoic acid, all-cis-9,12,15-octadecetrienoicacid, all-cis-5,8,11,14-eicosatetraenoic acid,all-cis-4,7,10,13,16,19-docosahexaenoic acid, 2,4,6,8-tetramethyldecanoic acid, and lactobacillic acid, and the like.

In some embodiments, pegylated lipid also is included. Pegylated lipidincludes lipids covalently linked to polymers of polyethylene glycol(PEG). PEG's are conventionally classified by their molecular weight,thus PEG 6,000 MW, e.g., has a molecular weight of about 6000. Addingpegylated lipid generally will result in an increase of the amount ofcompound (e.g., peptide, nucleotide, and nutrient) that can beincorporated into the precipitate. An exemplary pegylated lipid isdipalmitoylphosphatidylehtanolamine (DPPE) bearing PEG 5,000 MW.

The morpholino-cochleate compositions of the present invention can beprovided in a variety of forms (e.g. powder, liquid, suspension) with orwithout additional components. Suitable forms and additives, excipients,carriers and the like are described herein.

Aggregation Inhibitors

The cochleates and cochleate compositions of the present invention canoptionally include an aggregation inhibitor. Aggregation inhibitors workin part by modifying the surface characteristics of the cochleates suchthat aggregation is inhibited. Aggregation can be inhibited, forexample, by steric bulk and/or a change in the nature of the cochleatestructure, e.g., a change in the surface hydrophobicity and/or surfacecharge.

Aggregation can be inhibited and even reversed, and individual cochleateparticles can be stabilized by changing the surface properties of thecochleates and thereby inhibiting cochleate-cochleate interaction.Aggregation can be inhibited by including in the liposome suspension amaterial that prevents liposome-liposome interaction at the time ofcalcium addition and thereafter. Alternatively, the aggregationinhibitor can be added after formation of cochleates. Additionally, theamount of aggregation inhibitor can be varied, thus allowing modulationof the size of the cochleates.

In one embodiment, the aggregation inhibitor can be employed to achievecochleates that are significantly smaller and have narrower particlesize distributions than compositions without aggregation inhibitors.Such compositions are advantageous for several reasons including thatthey can allow for greater uptake by cells, and rapid efficacy. Such acomposition is suitable, e.g., when it is desired to rapidly andeffectively deliver encochleated molecules. Moreover, cochleate size canhave a targeting affect in that some cells may take up particles of acertain size more or less effectively. Size may also affect the mannerin which cochleates interact with a cell (e.g., fusion events oruptake).

In another embodiment, the aggregation inhibitor can be employed toachieve cochleate compositions having a particle size relatively largerthan that which can be achieved with or without aggregation inhibitors.Such a composition can be useful, e.g., when delayed uptake and/orrelease of encochleated molecules is desired, or when targeted cells ororgans more effectively take up cochleates in the relatively larger sizerange. Such compositions also may have sustained activity (relative tosmaller cochleate compositions) because it can take longer for themolecule to be released from a larger cochleate, e.g., if multiplefusion events are required.

In yet another embodiment, the cochleate composition has a wide particlesize distribution such that the encochleate molecule (e.g., siRNA ormorpholino and any additional cargo moeity) is released over a period oftime because smaller cochleates are rapidly taken up initially followedby take up or fusion events with increasingly larger cochleates. Inaddition, size may not only affect what type of cells take up thecochleates, but also how the cochleates interact with certain cells,e.g., size may effect whether a cochleate is taken up by a cell orundergoes one or more fusion events with a cell.

Moreover, in yet further embodiments, several compositions can becombined for desired release profiles, e.g., a pulsed released, orcombined release. For example, a rapid release nanocochleate compositioncan be mixed with a delayed-release larger size or even standardcochleate composition, such that an immediate and a delayed release isrealized. In addition, the cochleate compositions may have differentsiRNAs or morpholinos.

An aggregation inhibitor also can be employed to stabilize particle sizeand particle size distribution. For example, it can be used to “lock-in”the cochleate size and distribution of standard cochleates and/orcochleates having an aggregation inhibitor. While the cochleates of theinvention typically are stable over long periods of time, standardcochleates (cochleates formed without aggregation inhibitors) can tendto aggregate over time. Thus, standard cochleates can be stabilized byaddition to such aggregation inhibitors, e.g., addition ofmethylcellulose after cochleate formation.

Accordingly, in one embodiment, cochleate compositions of the inventionhave a mean diameter less than about 1 micrometer, e.g., less than about900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm,50 nm, 10 nm, or 1 nm. All individual values and ranges within theseranges are meant to be included and are within the scope of thisinvention. In another embodiment, cochleate compositions of theinvention have a mean diameter about equal to or greater than about 1micrometer, e.g., 2, 3, 4, 5, 10, 50, or 100 micrometers. All individualvalues and ranges within these ranges are meant to be included and arewithin the scope of this invention.

In one embodiment, the size distribution is narrow relative to thatobserved in standard cochleates (cochleates formed without aggregationinhibitors). Preferably, the cochleate compositions have a sizedistribution of less than about 1 μm, 900 nm, 800 nm, 700 nm, 600 nm,500 nm, 400 nm, 300 nm, 200 nm, or 100 nm. All individual values betweenthese values (550 nm, 420 nm, 475 nm, etc.), are meant to be includedand are within the scope of this invention. Such cochleate compositionsare particularly desirable where uptake by macrophages is desired. Itcan readily be appreciated that particle size can be adjusted to a sizesuitable for uptake by desired organs or cells and/or unsuitable foruptake by organs or cells. In another embodiment, a wider sizedistribution of cochleates is employed, e.g., about 10, 20, 50, 100,200, 300, 400 or 500 micrometers. All individual values within theseranges are meant to be included and are within the scope of thisinvention. Such cochleate compositions can be useful for long termrelease of cargo moieties.

Additionally, as discussed above, the invention contemplates combinationof cochleate populations to achieve a desired release pattern, e.g.,pulsed release and/or timed release of siRNAs or morpholinos against oneor more target mRNAs.

The type and/or amount of aggregation inhibitor used can also determinethe size of resulting cochleate. The presence of an aggregationinhibitor in differing concentrations also allows regulation ofcochleate size distribution.

Suitable aggregation inhibitors that can be employed in accordance withthe present invention, include but are not limited to at least one ofthe following: casein, K-casein, milk, albumin, serum albumin, bovineserum albumin, methylcellulose, ethylcellulose, propylcellulose,hydroxycellulose, hydroxymethyl cellulose, hydroxyethyl cellulose,hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, carboxymethyl cellulose, carboxyethyl cellulose, pullulan,polyvinyl alcohol, sodium alginate, polyethylene glycol, polyethyleneoxide, xanthan gum, tragacanth gum, guar gum, acacia gum, arabic gum,polyacrylic acid, methylmethacrylate copolymer, carboxyvinyl polymer,amylose, high amylose starch, hydroxypropylated high amylose starch,dextrin, pectin, chitin, chitosan, levan, elsinan, collagen, gelatin,zein, gluten, carrageenan, carnauba wax, shellac, latex polymers, milkprotein isolate, soy protein isolate, whey protein isolate and mixturesthereof.

A preferred aggregation inhibitor is casein. Casein is a highlyphosphorylated, calcium binding protein. Without wishing to be bound toany particular theory, it is believed that calcium mediates aninteraction between negatively charged lipid (e.g., PS) and casein,thereby changing the surface properties of cochleates such thataggregation is inhibited. Another preferred aggregation inhibitor ismilk and other milk products such as Half and Half, cream etc. Preferredmilk products also contain casein. Another preferred aggregationinhibitor is methylcellulose. In addition, more than one aggregationinhibitor may be employed in the cochleates of the invention. Forexample, both casein and methylcellulose may be used as an aggregationinhibitor.

In one embodiment, the cochleates of the invention include anaggregation inhibitor to lipid ratio of between about 0.1:1 to about 4:1by weight. Preferably, the aggregation inhibitor to lipid ratio is about1:1. A person of ordinary skill in the art will readily be able todetermine the amount of aggregation inhibitor needed to form cochleatesof the desired size with no more than routine experimentation.

Additional Cargo Moieties

The cochleates and cochleate compositions of the present invention canfurther include one or more additional cargo moieties. An “additionalcargo moiety” is an encochleated moiety in addition to the siRNA ormorpholino of the invention, and generally does not refer to the lipidand ion employed to precipitate the cochleate. Cargo moieties includeany compounds having a property of biological interest, e.g., ones thathave a role in the life processes of a living organism. A cargo moietymay be organic or inorganic, a monomer or a polymer, endogenous to ahost organism or not, naturally occurring or synthesized in vitro andthe like.

The cargo moiety can be a protonized cargo moiety. The term “protonizedcargo moiety” refers to a protonizable cargo moiety that has beenprotonized. “Protonizable” refers to the ability to gain one or moreprotons. The protonizable cargo moiety can be weakly basic, and can beprotonized by acidification or addition of a proton. Additionally oralternatively, the protonizable cargo moiety can be neutral or weaklyacidic and can be protonized in the same manner. Thus, the protonizablecargo moiety can be an anionic or a neutral cargo moiety, which isrendered cationic by protonization, or the protonizable cargo moiety canbe cationic, and be rendered more cationic upon protonization. The cargomoiety can also be provided protonized. Optionally, the protonized statecan be induced, e.g., by acidification or other methods, as describedherein. Acidification renders the cargo moiety cationic or increases thevalency of a cargo moiety that is already cationic, e.g., frommonovalent to divalent or trivalent.

Examplarary additional cargo moieties include vitamins, minerals,nutrients, micronutrients, amino acids, toxins, microbicides,microbistats, co-factors, enzymes, polypeptides, polypeptide aggregates,polynucleotides, lipids, carbohydrates, nucleotides, starches, pigments,fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids,flavorings, essential oils, extracts, hormones, cytokines, viruses,organelles, steroids and other multi-ring structures, saccharides,metals, metabolic poisons, imaging agents, antigens, porphyrins,tetrapyrrolic pigments, marker compounds, medicaments, drugs and thelike.

The cargo moiety can be a diagnostic agent, such as an imaging agent.Imaging agents include nuclear agents and fluorescent probes, e.g.,porphyrins. Porphyrins include tetrapyrrolic agents or pigments. Onesuch tetrapyrrolic agent is Zinc Tetra-Phenyl Porphyrin (ZnTPP), whichis a hydrophobic, fluorescent molecule that has high absorption in thevisible spectrum (dark purple).

The cargo moiety can be a polynucleotide that is expressed to yield abiologically active polypeptide or polynucleotide. Thus, the polypeptidemay serve as an immunogen or, e.g., have enzymatic activity. Thepolynucleotide may have catalytic activity, for example, be a ribosome,or may serve as an inhibitor of transcription or translation, e.g., asmall interfering RNA (siRNA) or an antisense molecule. Thepolynucleotide can be modified, e.g., it can be synthesized to have amorpholino backbone. If expressed, the polynucleotide preferablyincludes the necessary regulatory elements, such as a promoter, as knownin the art. A specific example of a polypeptide is insulin.

The drug can be an organic molecule that is hydrophobic in aqueousmedia. The drug can also be a water-soluble monovalent or polyvalentcationic molecule, anionic, or net neutral at physiological pH. The drugcan be, but is not limited to, a protein, a small peptide, a bioactivepolynucleotide, an antibiotic, an antiviral, an anesthetic, anantidepressant, an antipsychotic, an anti-infectious, an antifungal, ananticancer, an immunosuppressant, an immunostimulant, a steroidalanti-inflammatory, a non-steroidal anti-inflammatory, an antioxidant, anantidepressant which can be synthetically or naturally derived, asubstance which supports or enhances mental function or inhibits mentaldeterioration, an anticonvulsant, an HIV protease inhibitor, anon-nucleophilic reverse transcriptase inhibitor, a cytokine, atranquilizer, mucolytic agent, a dilator, a vasoconstrictor, adecongestant, a leukotriene inhibitor, an anti-cholinergic, ananti-histamine a cholesterol, a lipid metabolism modulating, or avasodilatory agent.

Examples of additional cargo moieties include Amphotericin B, acyclovir,adriamycin, carbamazepine, ivermectin, melphalen, nifedipine,indomethacin, curcumin, aspirin, ibuprofen, naproxen, acetaminophen,rofecoxib, diclofenac, ketoprofin, meloxicam, nabumetone, estrogens,testosterones, steroids, phenyloin, ergotamines, cannabinoids,rapamycin, propanadid, propofol, alphadione, echinomycin, miconazolenitrate, teniposide, hexamethylmelamine, taxol, taxotere,18-hydroxydeoxycorticosterone, prednisolone, dexamethazone, cortisone,hydrocortisone, piroxicam, diazepam, verapamil, vancomycin, tobramycin,teicoplanin, bleomycin, peptidolglycan, ristocetin, sialoglycoproteins,orienticin, avaporcin, helevecardin, galacardin, actinoidin, gentamycin,netilmicin, amikacin, kanamycin A, kanamycin B, neomycin, paromomycin,neamine, streptomycin, dihydrostreptomycin, apramycin, ribostamycin,spectinomycin, caspofungin, echinocandin B, aculeacin A, micafungin,anidulafungin, cilofungin, pneumocandin, geldanamycin, nystatin,rifampin, tyrphostin, a glucan synthesis inhibitor, vitamin A acid,mesalamine, risedronate, nitrofurantoin, dantrolene, etidronate,nicotine, amitriptyline, clomipramine, citalopram, dothepin, doxepin,fluoxetine, imipramine, lofepramine, mirtazapine, nortriptyline,paroxetine, reboxitine, sertraline, trazodone, venlafaxine, dopamine,St. John's wort, phosphatidylserine, phosphatidic acid, amastatin,antipain, bestatin, benzamidine, chymostatin, 3,4-dichloroisocoumarin,elastatinal, leupeptin, pepstatin, 1,10-phenanthroline, phosphoramidon,ethosuximide, ethotoin, felbamate, fosphenyloin, lamotrigine,levitiracetam, mephenyloin, methsuximide, oxcarbazepine, phenobarbital,phensuximide, primidone, topirimate, trimethadione, zonisamide,saquinavir, ritonavir, indinavir, nelfinavir, and amprenavir.

Additional drugs include, but are not limited to agents that reduce therisk of atherosclerotic events and/or complications thereof. Such agentsinclude, but are not limited to beta blockers, beta blockers andthiazide diuretic combinations, HMG CoA reductase inhibitors, statins,aspirin, ace inhibitors, ace receptor inhibitors (ARBs), and the like.Suitable beta blockers include, but are not limited to cardioselective(selective beta 1 blockers), e.g., acebutolol (e.g., Sectral™), atenolol(e.g., Tenormin™), betaxolol (e.g., Kerlone™), bisoprolol (e.g.,Zebeta™), metoprolol (e.g., Lopressor™), and the like. Suitablenon-selective blockers (block beta 1 and beta 2 equally) include, butare not limited to carteolol (e.g., Cartrol™), nadolol (e.g., Corgard™),penbutolol (e.g., Levatol™), pindolol (e.g., Visken™), propranolol(e.g., Inderal™), timolol (e.g., Blockadren™), labetalol (e.g.,Normodyne™, Trandate™), and the like.

Suitable beta blocker thiazide diuretic combinations include, but arenot limited to Lopressor HCT, ZIAC, Tenoretic, Corzide, Timolide,Inderal LA 40/25, Inderide, Normozide, and the like. Suitable statinsinclude, but are not limited to pravastatin (e.g., Pravachol™),simvastatin (e.g., Zocor™), lovastatin (e.g., Mevacor™), and the like.Suitable ace inhibitors include, but are not limited to captopril (e.g.,Capoten™), benazepril (e.g., Lotensin™), enalapril (e.g., Vasotec™),fosinopril (e.g., Monopril™), lisinopril (e.g., Prinivil™ or Zestril™),quinapril (e.g., Accupril™), ramipril (e.g., Altace™), imidapril,perindopril erbumine (e.g., Aceon™), trandolapril (e.g., Mavik™), andthe like. Suitable ARBS (Ace Receptor Blockers) include but are notlimited to losartan (e.g., Cozaar™), irbesartan (e.g., Avapro™),candesartan (e.g., Atacand™), valsartan (e.g., Diovan™), and the like.

Suitable HMG CoA reductase inhibitors that are useful in accordance withthe methods and compositions of the invention are statin molecules.These include: Lovastatin (e.g., Mevacor™), Pravastatin (e.g.,Pravachol™), Simvastatin (e.g., Zocor™), Fluvastatin (e.g., Lescol™),Atorvastatin (e.g., Lipitor™), or Cerivastatin (e.g., Baycol™).

Other agents that may be administered in conjunction with the cochleatesof the invention for treatment of atherosclerotic events and/orcomplications thereof are phytosterols, phytostanols and theirderivatives and isomers; soy protein; soluble fibers, e.g. beta-glucanfrom, for example, oat and psyllium, nuts, rice bran oil, each of whichis particularly suitable for use in food, dietary supplements and foodadditive compositions. Phytosterols may be solid (e.g., powder,granules) or liquid (e.g., oil) form.

It will be obvious to a person of skill in the art that the choice ofthe agent for treatment of atherosclerotic events and/or complicationsthereof depends on the intended delivery vehicle (e.g., food,supplement, pharmaceutical) and the mode of administration.

The cargo moiety can be a polypeptide such as cyclosporin, AngiotensinI, II and III, enkephalins and their analogs, ACTH, anti-inflammatorypeptides I, II, III, bradykinin, calcitonin, b-endorphin, dinorphin,leucokinin, leutinizing hormone releasing hormone (LHRH), insulin,neurokinins, somatostatin, substance P, thyroid releasing hormone (TRH)and vasopressin.

The cargo moiety can be an antigen, but is not limited to a proteinantigen. The antigen can also be a carbohydrate or DNA. Examples ofantigenic proteins include membrane proteins, envelope glycoproteinsfrom viruses, animal cell proteins, other viral proteins, plant cellproteins, bacterial proteins, and parasitic proteins.

Suitable nutrients include, but are not limited to lycopene,micronutrients such as phytochemicals or zoochemicals, vitamins,minerals, fatty acids, amino acids, fish oils, fish oil extracts,biotin, choline, inositol, ginko, and saccharides, herbal products oressential oils. Specific examples include Vitamins A, B, B1, B2, B3,B12, B6, B-complex, C, D, E, and K, vitamin precursors, caroteniods, andbeta-carotene, resveratrol, lutein, zeaxanthine, quercetin, silibinin,perillyl alcohol, genistein, sulfurophane, and essential fatty acids,including eicosapentanoic acid (EPA), gamma-3, omega-3, gamma-6, andomega-6 fatty acids, herbs, spices, and iron. Minerals include, but arenot limited to boron, chromium, colloidal minerals, colloidal silver,copper, manganese, potassium, selenium, vanadium, vanadyl sulfate,calcium, magnesium, barium, iron and zinc.

The cargo moiety can be a saccharide or sweetener, e.g., saccharine,isomalt, maltodextrine, aspartame, glucose, maltose, dextrose, fructoseand sucrose. Flavor agents include oils, essential oils, or extracts,including but not limited to oils and extracts of cinnamon, vanilla,almond, peppermint, spearmint, chamomile, geranium, ginger, grapefruit,hyssop, jasmine, lavender, lemon, lemongrass, marjoram, lime, nutmeg,orange, rosemary, sage, rose, thyme, anise, basil, and black pepper, teaor tea extracts, an herb, a citrus, a spice or a seed.

As used herein, the term “fragile nutrients” refers to fragile compounds(e.g., susceptible to degradation by oxygen, water and the like) derivedfrom plant sources (phytochemicals), animal sources (zoochemicals), orsynthetic sources that are either known or are suspected of contributingto the health of an animal.

As used herein, “micronutrient” is a nutrient that the body must obtainfrom outside sources. Generally micronutrients are essential to the bodyin small amounts.

In one embodiment, the cargo moiety is added to the composition in alipid to cargo moiety ratio from between about 20,000:1 to about 1:1.Preferably the cargo moiety is loaded in a lipid to cargo moiety ratiofrom about 10:1 to about 1:1. More preferably, the cargo moiety isloaded in a lipid to cargo moiety ratio of about 5:1. In anotherembodiment, a second cargo moiety is additionally incorporated into thecochleate structure in a lipid to cargo moiety ratio of between about20,000:1 to about 1:1. Preferably the second cargo moiety is loaded in alipid to cargo moiety ratio from about 10:1 to about 1:1. Morepreferably, the second cargo moiety is loaded in a lipid to cargo moietyratio of about 5:1.

Methods of Manufacture

In another aspect, the present invention generally is directed tomethods of making cochleates that include siRNA and/or morpholinos. Themethods generally can include precipitating a liposome suspension in thepresence of an siRNA component and/or a morpholino component, e.g., byadding a multivalent cation. The cochleates can further includeadditional cargo moieties or other constituents, e.g., aggregationinhibitors. All of the methods described herein can be employed formaking both morpholino-cochleates and siRNA cochleates.

Liposomes may be prepared by any known method of preparing liposomes.Thus, the liposomes may be prepared for example by solvent injection,lipid hydration, reverse evaporation, freeze drying by repeated freezingand thawing. The liposomes may be multilamellar or unilamellar,including small unilamellar vesicles (SUV). The liposomes may be largeunilamellar vesicles (LUV), stable plurilamellar vesicles (SPLV) oroligolamellar vesicles (OLV) prepared, e.g., by detergent removal usingdialysis, column chromatography, bio beads SM-2, by reverse phaseevaporation (REV), or by formation of intermediate size unilamellarvesicles by high pressure extrusion. Methods in Biochemical Analysis,33:337 (1988). Liposomes made by all these and other methods known inthe art can be used in practicing this invention.

In a preferred embodiment at least majority of the liposomes areunilamellar. The method can further include the step of filtering aliposomal suspension and/or mechanically extruding the suspensionthrough a small aperture that includes both MLV and ULV liposomes, suchthat a majority of the liposomes are ULV. In preferred embodiments, atleast 70%, 80%, 90% or 95% of the liposomes are ULV.

The method is not limited by the method of forming cochleates. Any knownmethod can be used to form cochleates from the liposomes of theinvention (i.e., the liposomes associated with the cargo moiety). In oneembodiment, known methods can be employed to form the cochleates of theinvention, including but not limited to those described in U.S. Pat.Nos. 5,994,318 and 6,153,217, the entire disclosures of which areincorporated herein by this reference.

In one embodiment, prior to precipitation, SUVs are obtained by, e.g.,filtration, and the liposomes are precipated in the presence of siRNA,morpholinos and/or other cargo moiety to form cochleates.

In another embodiment, MLVs are extruded one or more times in thepresence of siRNA, morpholinos and/or other cargo moiety, then theliposomes are precipated form cochleates. In this embodiment, it isbelieved that the MLVs open and reseal during the extrusion processthereby entrapping or otherwise increasing the association of the siRNA,morpholinos and/or other cargo moieties with the MLVs.

In yet another embodiment, a chelating agent (e.g., EDTA) is employed toconvert cochleates to liposomes in the presence of the siRNA ormorpholino and/or other cargo moiety, and then cation is added to formthe cochleates.

In yet another embodiment, enhanced binding of the siRNA or morpholinoand the liposome and/or cochleates is achieved by first forming acomplex between the siRNA or morpholino and a transfection agent. Such acationic transfection agent is preferably a polycation, e.g.,polyethylenimine (PEI), polyvinylamine, spermine, spermidine, histamine,cationic lipid, or other moiety to enhance binding to the liposome priorto precipitation. Alternatively the polycation is mixed with and bindsto the liposome first and then the RNA or morpholino is added. The hightransfection potential of DNA complexed with the cationic polymerpolyethylenimine (PEI) has been described. Boussif et al. Proc Natl AcadSci USA 92: 7297-7301 (1995). However, increased transfection rates havebeen coupled with increased toxicity. Bogden et al., AACS PharmSci 4(2)(2002). PEI can be obtained, e.g., from BASF, such as that sold underthe tradename Lupasol G35. Cationic polymers may be employed having avariety of molecular weights, and may be branched or unbranched.

It has been discovered, and illustrated the Examples provided herein,that encochleated siRNA-PEI complexes improve transfection into cellswithout the associated toxicity observed in the literature and in theExamples. In preferred embodiments the cation is a cationic polymer,e.g., PEI or PEI derivatives. Such complexes can be associated with theliposomes by any of the methods discussed herein.

The ratios of lipid to siRNA, and PEI to siRNA, etc. may vary. In apreferred embodiment, N to P ratios (N, nitrogen in PEI to P, phosphatein RNA) may vary from between about 0.5 to about 20. Most preferablybetween about 4 to about 8.

Additionally or alternatively, siRNA or morpholinos can be encochleatedwith high or low amounts of calcium. Accordingly, in one embodiment, ahigh or “elevated” amount of calcium is used, e.g., wherein the calciumconcentration in the solution when the cochleates are formed is betweenabout 100 and about 500 mMolar. As used herein, the term “elevatedamount of calcium” means a calcium concentration between about 100 andabout 500 mMolar. In another embodiment, a relatively low (“depressed”)amount of calcium is used, e.g., between about 1 to about 10 mM. As usedherein, the term “depressed amount of calcium” means a calciumconcentration between about 1 and about 10 mM. As demonstrated in theexamples, siRNA encochleated with high amounts of calcium were moreactive than siRNA encochleated with low amounts of calcium.

In one embodiment, the pH of the morpoholino or siRNA is adjusted inorder to induce a charge in the molecule and thereby increase itsinteraction with the cochleate, and in particular the phospholipid. Inone embodiment, the method includes adjusting the pH of the liposomalsuspension. In another embodiment, the method may include charging thebase pairs of the siRNA or morpholino. For example, the pH can beadjusted to about 8.5 or about 6.0 to 6.5 or about 3.0 to 3.5 for amorpholino. Raising the pH of a liposomal suspension in the presence ofmorpholino causes the morpholino to associate or complex with theliposomes. Raising or lowering the pH of the siRNA or morpholino(between 3 to 11) can affect charge on the bases or backbone and enhanceassociation with the lipid.

It has been discovered that adjusting the pH and/or charging the basepairs can improve association of the morpholino or siRNA with thecochleate. Accordingly, the method can further include the step ofadjusting the pH of the morpholino or siRNA prior to or during thecontact with the liposome or formation of the precipitate. Any knownmethod of adjusting pH can be employed. For example, a morpholino orsiRNA can be acidified with acidic aqueous buffer. Alternately, pH canbe raised with a basic aqueous buffer. Acidic and basic buffers areknown in the art, and identification of a variety of buffers wouldrequire no more than routine experimentation by one of ordinary skill inthe art. Alternatively, the pH of the morpholino or siRNA can beadjusted by slow addition of an acid, e.g., hydrochloric acid, or abase, e.g., sodium hydroxide.

In yet other embodiments, the pH of the morpholinos or siRNA can beadjusted prior to incorporation into the lipid precipitates. In otherembodiments, the pH of the resultant morpholino cochleates in solutioncan be adjusted using, e.g., acid or base.

In one embodiment, cochleates may be formed by dissolving a lipidcomponent and siRNA, morpholino and/or other cargo moiety in an organicsolvent (e.g., THF) to form a solution, forming cargo moiety liposomes,and precipitating the liposome to form a cargo moiety-cochleate. Thesolvent can optionally be removed prior to the formation of liposomesand/or after the liposomes are formed.

In another embodiment, cochleates can be formed by introducing themolecule (e.g, siRNA, morpholino and/or additional cargo moiety), to aliposome in the presence of a solvent such that the molecule associatewith the liposome, and precipitating the liposome to form a cochleate.The molecule can be introduced by introducing a solution of the solventand the molecule to the liposome by, e.g., dropwise addition, continuousflow or as a bolus. The molecule can also be introduced to the liposomeprior to or after the solvent.

The liposome may be prepared by any known method of preparing liposomes.Additionally, the method is not limited by the method of formingcochleates. Any known method can be used to form cochleates from theliposomes of the invention (i.e., the liposomes associated with thecargo moiety). In a preferred embodiment, the cochleate is formed byprecipitation. Additionally or alternatively, an aggregation inhibitorcan be added to the solvent at the liposomal stage, or to theprecipitated cochleate.

Any suitable solvent can be employed in connection with the presentinvention. Solvents suitable for a given application can be readilyidentified by a person of skill in the art. Suitable solvents includebut are not limited to dimethylsulfoxide (DMSO), a methylpyrrolidone,N-methylpyrrolidone (NMP), acetonitrile, alcohols, e.g., butanol andethanol (EtOH), dimethylformamide (DMF), tetrahydrofuran (THF), andcombinations thereof.

Moreover, the order of addition of various components (e.g., siRNA,lipid, calium, cation complexing agents, solvent) can readily be variedas exemplified in the Examples provided in the instant application.Concentrations and ratios of various components can also be modified asexemplified herein. Finally, ionic conditions may be adjusted asappropriate. Salt concentrations may be approximately isotonic (150 mM),to high (e.g., 1 to 2 molar), to hypotonic, to zero (water).

An exemplary method of forming morpholino-cochleates in accordance withthe present invention can generally include the following steps.Liposomes and morpholino oligonucleotides can be solubilized andvortexed to form a morpholino-liposome suspension. Typically, about 2minutes of vortexing is sufficient to form a suitable suspension, whichcan be varied and confirmed by visual inspection, e.g., through amicroscope.

Next, the pH of the suspension is either raised to about 8.5 (e.g., with1 N NaOH) or lowered to about 6.5 (e.g., with 1 N HCl). Since themorpholinos are non-charged, this step is done to place a charge on thebase pairs of the morpholino, to favor an interaction with theliposomes. This ionic interaction can be achieved by either increasingthe pH to 8.5 or by lowering the pH to 6.5. At this point themorpholinos interact with the lipid. The suspension is again vortexed toinduce interaction between the morpholinos and the liposomes. Typically,about 10 minutes of vortexing is suitable. Interaction between themorpholinos and the liposomes can be confirmed by phase and defractionmicroscopy. The morpholinos associate with or incorporate into theliposomal bilayer. The morpholino-liposomes are then filtered (e.g.,using a 0.22 micrometer syringe filter).

Calcium solution is added to the suspension with vortexing. A suitableaddition technique is to use an eppendorf repeater pipetter with a 500microliter tip, and to add 10 microliter aliquots to the tube every 10seconds until cochleates are formed. Cochleate formation can beconfirmed, e.g., by observing the preparation under a microscope and bya measurement of pH. The cochleates can then be stably stored at 4° C.in a nitrogen atmosphere.

Methods of Use

In another aspect, the invention provides methods of administering siRNAor morpholinos to a host (e.g. a cell or organism). The method generallyincludes administering a biologically effective amount of asiRNA-cochleate or morpholino-cochleate composition to a host. Thecochleate compositions can include any of the compositions describedherein including, e.g., compositions with additional cargo moietiesand/or aggregation inhibitors.

The host can be a cell, a cell culture, an organ, a tissue, andorganism, an animal etc. For example, in one embodiment, the siRNA ormorpholino is delivered to a cell in the host (e.g., to a cytosolcompartment of the cell).

In one embodiment the siRNA mediates RNAi against a target mRNA in thehost. In another embodiments, the morpholino mediates translation of atarget mRNA in the host. In either embodiment, although acting by adifferent mechanism, specific target protein synthesis preferably isreduced in the host. In preferred embodiments, target protein synthesisis reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or even 100%.

Physical methods of introducing siRNAs and morpholinos to cells andorganisms employing cochleates include contacting the cells with thecochleates or administering the cochleates to the organism by any means,e.g., orally, intramuscularly, intradermally, transdermally,intranasally, intrarectally, subcutaneously, topically, orintravenously.

siRNA-cochleates and morpholino-cochleates may be directly introduced toor into a cell (i.e., intracellularly), or introduced extracellularlyinto a cavity, interstitial space, into the circulation of an organism,introduced orally, or may be introduced by bathing a cell or organism ina solution containing the cochleates. Vascular or extravascularcirculation, the blood or lymph system, and the cerebrospinal fluid aresites where the cochleate compositions of the present invention may beintroduced.

One mechanism by which the siRNA, morpholinos and/or other cargomoieties may be introduced to a cell is via a fusion event between thecochleate and the cell. Many naturally occurring membrane fusion eventsinvolve the interaction of calcium with negatively charged phospholipids(e.g., PS and phosphatidylglycerol). Calcium-induced perturbations ofmembranes containing negatively charged lipids, and the subsequentmembrane fusion events, are important mechanisms in many naturalmembrane fusion processes. Therefore, cochleates can be envisioned asmembrane fusion intermediates. As the calcium rich, highly orderedmembrane of a cochleate first comes into close approximation to anatural membrane, a perturbation and reordering of the cell membrane isinduced, resulting in a fusion event between the outer layer of thecochleate and the cell membrane. This fusion results in the delivery ofa small amount of the material associated with the cochleate into thecytoplasm of the target cell. The cochleate can then break free of thecell and be available for another fusion event, either with the same oranother cell.

Additionally or alternatively, particularly with active phagocyticcells, cochleates may be taken up by endocytosis and fuse from withinthe endocytic vesicle. Cochleates made with trace amounts of fluorescentlipids have been shown to bind and gradually transfer lipids to theplasma membrane and interior membranes of white blood cells in vitro.Accordingly, the encochleated siRNA, morpholinos and/or additional cargomoieties of the invention can be introduced to a cell in a host byendocytosis. Alternatively they may be introduced by pinocytosis.

A cell or tissue with a target mRNA may be derived from or contained inany organism. The organism may be a plant, animal, protozoan, bacterium,virus, or fungus. The plant may be a monocot, dicot or gymnosperm; theanimal may be a vertebrate or invertebrate. Preferred microbes are thoseused in agriculture or by industry, and those that are pathogenic forplants or animals. Fungi include organisms in both the mold and yeastmorphologies. Plants include arabidopsis; field crops (e.g., alfalfa,barley, bean, corn, cotton, flax, pea, rape, nice, rye, safflower,sorghum, soybean, sunflower, tobacco, and wheat); vegetable crops (e.g.,asparagus, beet, broccoli, cabbage, carrot, cauliflower, celery,cucumber, eggplant, lettuce, onion, pepper, potato, pumpkin, radish,spinach, squash, taro, tomato, and zucchini); fruit and nut crops (e.g.,almond, apple, apricot, banana, black-berry, blueberry, cacao, cherry,coconut, cranberry, date, filbert, grape, grapefruit, guava, kiwi,lemon, lime, mango, melon, nectarine, orange, papaya, passion fruit,peach, peanut, pear, pineapple, pistachio, plum, raspberry, strawberry,tangerine, walnut, and watermelon); and ornamentals (e.g., alder, ash,aspen, azalea, birch, boxwood, camellia, carnation, chrysanthemum, elm,fir, ivy, jasmine, juniper, oak, palm, poplar, pine, redwood,rhododendron, rose, and rubber). Examples of vertebrate animals includefish, mammal, cattle, goat, pig, sheep, rodent, hamster, mouse, rat,primate, and human; invertebrate animals include nematodes, other worms,drosophila, and other insects.

The cell having the target mRNA may be from the germ line or somatic,totipotent or pluripotent, dividing or non-dividing, parenchyma orepithelium, immortalized or transformed, or the like. The cell may be astem cell or a differentiated cell. Cell types that are differentiatedinclude adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium,neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages,neutrophils, eosinophils, basophils, mast cells, leukocytes,granulocytes, keratinocytes, chondrocytes, ostcoblasts, osteoclasts,hepatocytes, and cells of the endocrine or exocrine glands.

Depending on the particular target mRNA and the dose of siRNA and/ormorpholino material delivered, this process may provide partial orcomplete loss of function for the target mRNA in a host. A reduction orloss of mRNA expression in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95% or 99% or more of the host or targeted cells in the host isexemplary. Inhibition of mRNA expression refers to the absence (orobservable decrease) in the level of protein and/or mRNA product from atarget mRNA. Specificity refers to the ability to inhibit the targetmRNA without manifest effects on other genes of the cell. Theconsequences of inhibition can be confirmed by examination of theoutward properties of the cell or organism (as presented below in theexamples) or by biochemical techniques such as RNA solutionhybridization, nuclease protection, Northern hybridization, reversetranscription, gene expression monitoring with a microarray, antibodybinding, enzyme linked immunosorbent assay (ELISA), Western blotting,radioimmunoassay (RIA), other immunoassays, and fluorescence activatedcell analysis (FACS).

A simple assay that can be employed for assessing siRNA delivery andactivity in a variety of compositions follows, many variations on whichcan readily be ascertained by the skilled practitioner. anti-GFP siRNAand non-specific siRNA can be obtained from commercial sources, e.g.,Ambion, Inc (Austin, Tx). Anti-GFP siRNA and non-specific siRNAcompositions, e.g., various cochleate compositions described herein orknown in the art can be manufactured. Cells, such as SKOV cells can betransfected with green fluorescent protein (GFP) expressing plasmid,followed by treatment with anti-GFP siRNA and non-specific siRNAcompositions and any other suitable controls. GFP fluorescence can thenbe measured after a predetermined time period, e.g., 48 or 72 hours.This data can then be compared to determine which compositions were moreeffective at delivery of active siRNA than others.

For RNA-mediated inhibition in a cell line or whole organism, mRNAexpression is conveniently assayed by use of a reporter or drugresistance mRNA whose protein product is easily assayed. Such reportergenes include acetohydroxyacid synthase (AHAS), alkaline phosphatase(AP), beta galactosidase (LacZ), beta glucoronidase (GUS),chloramphenicol acetyltransferase (CAT), green fluorescent protein(GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase(NOS), octopine synthase (OCS), and derivatives thereof. Multipleselectable markers are available that confer resistance to ampicillin,bleomycin, chloramphenicol, gentarnycin, hygromycin, kanamycin,lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin.Depending on the assay, quantitation of the amount of mRNA expressionallows one to determine a degree of inhibition which is greater than10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not treatedaccording to the present invention. Lower doses of injected material andlonger times after administration of encochleated siRNA and/ormorpholinos may result in inhibition in a smaller fraction of cells(e.g., at least 10%, 20%, 50%, 75%, 90%, or 95% of targeted cells).Quantitation of mRNA expression in a cell may show similar amounts ofinhibition at the level of accumulation of target mRNA or translation oftarget protein.

As an example, the efficiency of inhibition may be determined byassessing the amount of mRNA product in the cell; mRNA may be detectedwith a hybridization probe having a nucleotide sequence outside theregion used for the inhibitory double-stranded RNA, or translatedpolypeptide may be detected with an antibody raised against thepolypeptide sequence of that region.

The RNA may be introduced in an amount which allows delivery of at leastone copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000copies per cell) of material may yield more effective inhibition; lowerdoses may also be useful for specific applications.

The cochleates can be coadministered with a further agent. The secondagent can be delivered in the same cochleate preparation, in a separatecochleate preparation mixed with the cochleates preparation of theinvention, separately in another form (e.g., capsules or pills), or in acarrier with the cochleate preparation. The cochleates can furtherinclude one or more additional cargo moieties, such as other drugs,peptides, nucleotides (e.g., DNA and RNA), antigens, nutrients, flavorsand/or proteins. Such molecules have been described in U.S. Pat. Nos.6,153,217 (Jin et al.) and 5,994,318 (Gould-Fogerite et al.), andInternational Patent Publication Nos. WO 00/42989 (Zarif et al.) and WO01/52817 (Zarif et al.). These patents are expressly incorporated bythis reference.

The cochleates of the invention also can include a reporter molecule foruse in in vitro diagnostic assays, which can be a fluorophore,radiolabel or imaging agent. The cochleates can include molecules thatdirect binding of the cochleate to a specific cellular target, orpromotes selective entry into a particular cell type.

Another advantage of the present invention is the ability to modulatecochleate size. Modulation of the size of cochleates can change themanner in which the siRNA, morpholino and/or additional cargo moiety istaken up by cells. For example, in general, small cochleates are takenup quickly and efficiently into cells, whereas larger cochleates aretaken up more slowly, but tend to retain efficacy for a longer period oftime. Also, in some cases small cochleates are more effective than largecochleates in certain cells, while in other cells large cochleates aremore effective than small cochleates.

Methods of Treatment

In another aspect, the present invention provides for both prophylacticand therapeutic methods of treating a subject at risk of (or susceptibleto) a disorder or having a disorder associated with aberrant or unwantedtarget gene expression or activity. The method generally includesadministering to a subject a therapeutically effective amount of amorpholino-cochleate composition or siRNA-cochleate of the inventionsuch that the disease or disorder is treated.

The present invention provides a method for treating a subject thatwould benefit from administration of a composition of the presentinvention. Any therapeutic indication that would benefit from thecochleate compositions of the present invention can be treated by themethods of the invention. The method includes the step of administeringto the subject a composition of the invention, such that the disease ordisorder is treated.

With regard to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics,” as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype,” or “drug response genotype”). Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the target genemolecules of the present invention or target gene modulators accordingto that individual's drug response genotype. Pharmacogenomics allows aclinician or physician to target prophylactic or therapeutic treatmentsto patients who will most benefit from the treatment and to avoidtreatment of patients who will experience toxic drug-related sideeffects.

The language “therapeutically effective amount” is that amount necessaryor sufficient to produce a desired physiologic response. The effectiveamount may vary depending on such factors as the size and weight of thesubject, or the particular compound. The effective amount may bedetermined through consideration of the toxicity and therapeuticefficacy of the compounds by standard pharmaceutical procedures in cellcultures or experimental animals, e.g., for determining the LD₅₀ (thedose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itmay be expressed as the ratio LD₅₀/ED₅₀. Compounds which exhibit largetherapeutic indices are preferred. While compounds that exhibit toxicside effects may be used, care should be taken to design a deliverysystem that targets such compounds to the site of affected tissue inorder to minimize potential damage to unaffected cells and, thereby,reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compositionused in the method of the invention, the therapeutically effective dosecan be estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the EC₅₀ (i.e., the concentration ofthe test composition that achieves a half-maximal response) asdetermined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant or unwantedtarget gene expression or activity, by administering to the subject atherapeutic agent (e.g., morpholinos, siRNAs or vector or transgeneencoding same). Administration of a prophylactic agent can occur priorto the manifestation of symptoms characteristic of the target geneaberrancy, such that a disease or disorder is prevented or,alternatively, delayed in its progression. Depending on the type oftarget gene aberrancy, for example, a target gene, target gene agonistor target gene antagonist agent can be used for treating the subject.The appropriate agent can be determined based on screening assaysdescribed herein.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulating targetgene expression, protein expression or activity for therapeuticpurposes. Accordingly, in an exemplary embodiment, the modulatory methodof the invention involves administering to a host a composition of theinvention that is specific for the target gene or protein (e.g., isspecific for the mRNA encoded by said gene or specifying the amino acidsequence of said protein) such that expression or one or more of theactivities of target protein is modulated. These modulatory methods canbe performed in vivo (e.g., by culturing the cell with the agent) or,alternatively, in vivo (e.g., by administering the agent to a subject).As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant or unwanted expression or activity of a target mRNA polypeptideor nucleic acid molecule. Inhibition of target mRNA activity isdesirable in situations in which the target mRNA is abnormallyunregulated and/or in which decreased target mRNA activity is likely tohave a beneficial effect.

One advantage of the cochleates of the present invention is the safetyand stability of the composition. Cochleates can be administered orallyor by instillation without concern, as well as by the more traditionalroutes, such as oral, intranasal, intraoculate, intraanal, intravaginal,intrapulmonary, topical, subcutaneous, intradermal, intramuscular,intravenous, subcutaneous, transdermal, systemic, intrathecal (intoCSF), and the like. Direct application to mucosal surfaces is anattractive delivery means made possible with cochleates.

The disease or disorder treated in accordance with the present inventioncan be any disease or disorder that can be treated by the successfuladministration of siRNAs or morpholinos of the invention. Exemplarydiseases and disorders include neurological disorders associated withaberrant or unwanted gene expression such as schizophrenia, obsessivecompulsive disorder (OCD), depression and bipolar disorder, Alzheimer'sdisease, Parkinson's disease, lymphoma, immune-mediated inflammatorydisorders, hyperplasia, cancers, cell proliferative disorders, bloodcoagulation disorders, Dysfibrinogenaemia and hemophelia (A and B),dematological disorders, hyperlipidemia, hyperglycemia,hypercholesterolemia, obesity, acute and chronic leukemias andlymphomas, sarcomas, adenomas, fungal infections, bacterial infections,viral infections, a lysosomal storage disease, Fabry's disease,Gaucher's Disease, Type I Gaucher's Disease, Farber's disease,Niemann-Pick disease (types A and B), globoid cell leukodystrophy(Krabbe's disease), metachromic leukodystrophy, multiple sulfatasedeficiency, sulfatidase activator (sap-B) deficiency, sap-C deficiency,G_(M1)-gangliosidosis, Tay-Sachs disease, Tay-Sachs B1 variant,Tay-Sachs AB variant, Acid Maltase Deficiency, Mucopolysaccharidosis,Sandhoff's disease, a cancer, an autoimmune disorder, systemic lupuserythematosis, multiple sclerosis, myasthenia gravis, autoimmunehemolytic anemia, autoimmune thrombocytopenia, Grave's disease,allogenic transplant rejection, rheumatoid arthritis, ankylosingspondylitis, psoriasis, scleroderma, carcinomas, epithelial cancers,small cell lung cancer, non-small cell lung cancer, prostate cancer,breast cancer, pancreatic cancer, hepatocellular carcinoma, renal cellcarcinoma, biliary cancer, colorectal cancer, ovarian cancer, uterinecancer, melanoma, cervical cancer, testicular cancer, esophageal cancer,gastric cancer, mesothelioma, glioma, glioblastoma, pituitary adenomas,inflammatory diseases, osteoarthritis, atherosclerosis, inflammatorybowel diseases (Crohns and ulcerative colitis), uveitis, eczema, chronicrhinosinusitis, asthma, a hereditary disease, cystic fibrosis, andmuscular dystrophy.

The method can also be used for regulating gene expression to promotegreater health or quality of life, e.g., to limit cholesterol uptake orregulate lipid metabolism, weight gain, hunger, aging, or growth.Cosmetic effects such as wrinkle reduction, hair growth, pigmentation,or dermatologic disorders may also be treated.

The compositions of the present invention can be used to enhanceantiviral defense, transposon silencing, gene regulation, centromericsilencing, and genomic rearrangements. The compositions of the inventioncan also be used to inhibit expression of other types of RNA, e.g.,ribosomal RNA, transfer RNA, and small nuclear RNA.

The siRNA and morpholino cochleate compositions of the present inventioncan be utilized in any number of gene therapies. One such treatment isfor the management of opportunistic fungal infections like Aspergillusfumigatus, particularly in immunocompromised patients. Current treatmentprotocols with existing antifungal agents can still result in mortalityrates of 80% in HIV patients or those undergoing cancer-relatedchemotherapies. However, the targeted disruption of the P-typeH+-ATPase, an important plasma membrane enzyme critical to fungal cellphysiology, may be an alternate and more effective way to destroy fungisuch as A. fumigatus. This particular ATPase was cloned and selectivesmall interfering RNA (siRNA) oligonucleotides obtained, which canknockdown the expression of this critical protein, resulting in thedeath of the fungus. siRNA-cochleates having siRNA targeted to theH+-ATPase of A. fumigatus will be delivered using cochleate compositionsas described herein.

The essential role of the H+-ATPase in spore germination andmultiplication of growing cells provides an opportunity to explore theability of nanocochleates to efficiently deliver siRNAs targeted to theH+-ATPase of A. fumigatus. Given the medical importance of A. fumigatusand the paucity of available antifungal compounds, siRNA cochleatecompositions have the potential to be effective therapeuticalternatives.

Treatment of Fungal Infections

Opportunistic fungal infections are widespread in cancer, HIV infectedand other immunosuppressed individuals, and are a growing concern forthe management of such patients. These organisms have become importantcauses of morbidity and mortality in the immunocompromised (Jarvis, W.R., (1995) Clin Infect Dis. 20(6): 1526-30; Dupont Jarvis, B., et al.,(1994) J Med Vet Mycol 32(Suppl 1): 65-77; Bodey, G. P. (1988) J HospInfect 11 Suppl A:411-26), and make opportunistic fungal infections amajor source of nosocomial disease. Pfaller, M. A. (1995) Clin InfectDis. 20(6):1525; Pfaller, M. A., et al., (1999) Diagn Microbiol InfectDis, 33(4): p. 217-22; Pfaller, M. A., et al., (1998) Diagn MicrobiolInfect Dis 31(1): 289-96; Pfaller, M. A., et al., (1998) Diagn MicrobiolInfect Dis 30(2):121-9; Pfaller, M. A., et al., (1998) J Clin Microbiol,36(7): 1886-9. The mold Aspergillus fumigatus causes a variety ofdiseases including allergic bronchopulmonary aspergillosis in asthmapatients and invasive pulmonary aspergillosis (IPA) in immunocompromisedpatients. Denning, D. W. (1998) Clin Infect Dis 26(4): 781-803; quiz804-5; Andriole, V. T. (1993) Clin Infect Dis. 17 Suppl 2: S481-6;Latge, J. P. (1999) Clin Microbiol Rev. 12(2): 310-50. Invasiveaspergillosis is a common infection in patients who areimmunocompromised, particularly in oncology patients, patients receivingother immunosuppressive therapy, bone marrow transplant patients, andHIV-infected patients. Aspergillus fumigatus accounts for 30% of fungalinfections among cancer patients and 10 to 25% in leukemia patients(Denning, D. W. (1998) Clin Infect Dis 26(4): 781-803; quiz 804-5).Early diagnosis of invasive fungal infections is critical for successfultherapeutic outcome, although such determinations are difficult toachieve. Denning, D. W. (1996) Curr Clin Top Infect Dis. 16: 277-99;Andriole, V. T., (1996) Infect Agents Dis. 5(1): 47-54; Latge, J. P.,(1995) Curr Top Med Mycol. 6: 245-81. The spectrum of diseasemanifestations is determined by a combination of genetic predisposition,host immune system defects, and virulence of the Aspergillus species.Amphotericin B is the standard of treatment for severe Aspergillusinfections (Stevens, D. A. et al., (2000) Clin Infect Dis. 30(4):696-709.), although mortality in these patients remains high. Latge, J.P., (1999) Microbiol Rev. 12(2): 310-50. In addition, amphotericin B maycause toxicity resulting in severe side effects, including permanentrenal insufficiency. Newer compositions, like liposomal suspensions, canreduce toxicity but do not eliminate it. Stevens, D. A. et al., (2000)Clin Infect Dis. 30(4): 696-709; Groll, A. H., et al., (1998) KlinPadiatr, 210(4): 264-73; Leenders, A. C., et al., (1998) Br J Haematol,103(1): 205-12; Ng, T. T. et al. (1995) Arch Intern Med. 155(10):1093-8; Robinson, R. F. et al. (1999) J Clin Pharm Ther. 24(4): 249-57.As the incidence of topical and invasive mycoses increases, there is acontinuing need to develop more effective therapeutics to deal withopportunistic fungal infections and to better understand thepathogenicity of these organisms.

Plasma Membrane Proton Pump (H⁺-ATPase)

The plasma membrane of all fungi contains an essential proton pumpingATPase (H⁺-ATPase) that regulates intracellular pH (Morsomme, P. et al.(2000) Biochim Biophys Acta. 1469(3): 133-57; Serrano, R., (1998)Biochim. Biophys. Acta. 947: 1-28), and maintains the electrochemicalproton gradient across the plasma membrane, which is necessary fornutrient uptake, including certain essential amino acids, sugars andions. Serrano, R., (1998) Biochim. Biophys. Acta. 947: 1-28. The plasmamembrane H⁺-ATPase has been extensively studied at the biochemical,biophysical and genetic levels (Morsomme, P. et al. (2000) BiochimBiophys Acta. 1469(3): 133-57; Perlin, D. S., et al. (1992) Acta PhysiolScand Suppl. 607: 183-92; Moller, J. V. et al. (1996) Biochim. Biophys.Acta. 1286: 1-51.) in model organisms such as Saccharomyces cerevisiae.It consists of a single M_(r)˜100 kDa polypeptide that is a predominantmembrane constituent representing 5-30% of the total membrane protein.Monk, B. C., et al., (1991) J Bacteriol. 173(21): 6826-36. It utilizesenergy from ATP hydrolysis to actively pump protons from inside the cellto the outside. The H⁺-ATPase is a typical Class IIIa P-type iontranslocating ATPase that includes the Na⁺, K⁺-ATPase of animal cellplasma membranes, Ca²⁺-ATPases of sarcoplasmic reticulum and red bloodcells.

The consensus view of the topology and secondary-structure model forH⁺-ATPase and other type II P-type ATPases enzyme it that they areorganized into three distinct structure-function domains. Zhang, P. etal. (1998) Nature. 392: 835-839. The cytoplasmic domain contains thesites for ATP binding and phosphorylation. The membrane embeddedtransport region contains ten α-helical transmembrane segments.Transmembrane segments M4, M5 and M6 contain aspartate and glumatateresidues important for cation binding and ion translocation. Lutsenko,S. et al. (1995) Biochemistry. 34(48): 15607-13.; MacLennan, D. H et al.(1997) J Biol Chem. 272(46): 28815-8.

The gene encoding the fungal H⁺-ATPase, PMA1, shows extensive amino acidsequence similarity between the various fungal enzymes (51-98%), butless than 30% similarity with its animal cell counterparts. Wach, A., A.et al. (1992) J. Bioenerg. Biomembr., 24: 309-317. The catalytic ATPhydrolysis domain displays the highest level of conservation, althoughsignature sequences are found outside this region as well. Lutsenko, S.et al. (1995) Biochemistry. 34(48): 15607-13. The N and C-termini of theP-type ATPases are highly divergent, as are extracellular loop domainslinking transmembrane segments, which contribute regulatory features ofeach class of ATPase. The divergence of structure on the extracellularface of the bilayer occurs among P-type ATPases with different ionspecificities but also in isoforms. It accounts for the differentialresponse of the animal cell Na⁺, K⁺-ATPase to cardiac glycosides(Lingrel, J. B. et al. (1994) J. Biol. Chem. 269: 19659-19662) and forthe specificity of antiulcer drugs like omeprazole to the gastric H⁺,K⁺-ATPase. Sachs, G., et al. (1995) Annu. Rev. Pharmacol. Toxicol., 35:277-305. It is this well documented ability to develop drug specificitybetween P-type ATPase molecules that has contributed to the success ofthis enzyme family as therapeutic targets, and could facilitate thedevelopment of highly specific antifungal drugs.

The plasma membrane H⁺-ATPase plays a critical role in fungal cellphysiology and it is one of the few antifungal targets that have beendemonstrated to be essential by gene disruption. Serrano, R. et al.(1986) Nature, 1986. 319: 689-693. The fungal H⁺-ATPase has attributesthat are attractive as a drug discovery target. It is an essentialenzyme that is needed for both new growth and stable cell maintenance inthe absence of growth. Due to its slow turnover in the membrane (˜11 h),it is likely that inhibitors of the H⁺-ATPase will be fungicidal.Preliminary studies with Ebselen, a model compound that inhibits ATPhydrolysis illustrates its fungicidal properties in Cryptococcusneoformans. Soteropoulos, P., et al. (2000) Antimicrob Agents Chemother.44(9): 2349-55. Several recent reports demonstrate H⁺-ATPase-mediatedantifungal properties from novel reagents including CAN-296, a complexcarbohydrate (Ben-Josef, A. M., et al. (2000) Int J Antimicrob Agents.13(4): 287-95) and NC1175,(3-[3-(4-chlorophenyl)-2-propenoyl]-4-[2-(4-chlorophenyl)vinylene]-1-ethyl-4-piperidinolhydrochloride) a thiol-blocking conjugated styryl ketone that alsoexhibits antifungal activity against a wide spectrum of pathogenicfungi. Manavathu, E. K., et al. (1999) Antimicrob Agents Chemother,43(12): 2950-9.

The essential role of the P type, H+-ATPase in fungal cell physiologymakes this enzyme a good target model for the efficacy of cochleatenanotechnology to deliver the cochleates of the present invention. Giventhe medical importance of Aspergillus fumigatus infection inimmunocompromised individuals, and the paucity of available antifungalcompounds, siRNA cochleates have the potential to be an effectivetherapeutic alternative.

Combination Therapies

The above methods can be employed in the absence of other treatment, orin combination with other treatments. Such treatments can be startedprior to, concurrent with, or after the administration of thecompositions of the instant invention. Accordingly, the methods of theinvention can further include the step of administering a secondtreatment, such as a second treatment for the disease or disorder or toameliorate side effects of other treatments. Such second treatment caninclude, e.g., any treatment directed toward reducing an immuneresponse. Additionally or alternatively, further treatment can includeadministration of drugs to further treat the disease or to treat a sideeffect of the disease or other treatments (e.g., anti-nausea drugs).

In one aspect, the invention provides a method for preventing in asubject, a disease or disorder which can be treated with administrationof the compositions of the invention. Subjects at risk for a disease orcondition which can be treated with the agents mentioned herein can beidentified by, for example, any or a combination of diagnostic orprognostic assays known to those skilled in the art. Administration of aprophylactic agent can occur prior to the manifestation of symptomscharacteristic of the disease or disorder, such that the disease ordisorder is prevented or, alternatively, delayed in its progression.

Pharmaceutical Compositions

The invention pertains to uses of the cochleates of the invention forprophylactic and therapeutic treatments as described infra. Accordingly,the cochleates of the present invention can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically comprise the cochleates of the invention and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.

Cochleates of the present invention readily can be prepared from safe,simple, well-defined, naturally occurring substances, e.g.,phosphatidylserine (PS) and calcium. Phosphatidylserine is a naturalcomponent of all biological membranes, and is most concentrated in thebrain. The phospholipids used can be produced synthetically, or preparedfrom natural sources. Soy PS is inexpensive, available in largequantities and suitable for use in humans. Additionally, clinicalstudies indicate that PS is safe and may play a role in the support ofmental functions in the aging brain. Unlike many cationic lipids,cochleates (which are composed of anionic lipids) are non-inflammatoryand biodegradable. The tolerance in vivo of mice to multipleadministrations of cochleates by various routes, including intravenous,intraperitoneal, intranasal and oral, has been evaluated. Multipleadministrations of high doses of cochleate compositions to the sameanimal show no toxicity, and do not result in either the development ofan immune response to the cochleate matrix, or any side effects relatingto the cochleate vehicle.

The cochleates of the present invention can be administered to animals,including both human and non-human animals. It can be administered toanimals, e.g., in animal feed or water.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, sweetening, flavoring and perfuming agents, preservatives andantioxidants may also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants, which may also bepresent in compositions of therapeutic compounds of the invention,include water soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and metalchelating agents, such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Compositions of the present invention include those suitable for oral,nasal, topical, transdermal, buccal, sublingual, rectal, vaginal orparenteral administration. The compositions may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which maybe combined with a carrier material to produce a single dosage form willgenerally be that amount of the composition which produces a therapeuticeffect. Generally, out of 100%, this amount will range from about 1% toabout 99% of active ingredient, preferably from about 5% to about 70%,most preferably from about 10% to about 30%.

Methods of preparing these compositions or compositions include the stepof bringing into association a composition of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the compositions are prepared by uniformly and intimatelybringing into association a composition of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Compositions of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) or as mouth washes and the like,each containing a predetermined amount of a composition of the presentinvention as an active ingredient. A composition of the presentinvention may also be administered as a bolus, electuary, or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules, and the like),the cochleates of the present invention are mixed with one or morepharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate, or any of the following: fillers or extenders, suchas starches, lactose, sucrose, glucose, mannitol, or silicic acid;binders, such as, for example, carboxymethylcellulose, alginates,gelatin, polyvinyl pyrrolidone, sucrose or acacia; humectants, such asglycerol; disintegrating agents, such as agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; solution retarding agents, such as paraffin; absorptionaccelerators, such as quaternary ammonium compounds; wetting agents,such as, for example, cetyl alcohol and glycerol monostearate;absorbents, such as kaolin and bentonite clay; lubricants, such a talc,calcium stearate, magnesium stearate, solid polyethylene glycols, sodiumlauryl sulfate, and mixtures thereof; and coloring agents.

In the case of capsules, tablets and pills, the pharmaceuticalcompositions may also comprise buffering agents. Solid compositions of asimilar type may also be employed as fillers in soft and hard-filledgelatin capsules using such excipients as lactose or milk sugars, aswell as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compositionmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes or microspheres.

They may be sterilized by, for example, filtration through abacteria-retaining filter, or by incorporating sterilizing agents in theform of sterile solid compositions which may be dissolved in sterilewater, or some other sterile injectable medium immediately before use.

These compositions may also optionally contain opacifying agents and maybe of a composition that they release the active ingredient(s) only, orpreferentially, in a certain portion of the gastrointestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich may be used include polymeric substances and waxes. The activeingredient may also be in micro-encapsulated form, if appropriate, withone or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof. Besides inert diluents, theoral compositions may also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, coloring,perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Compositions of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound. Compositions of thepresent invention which are suitable for vaginal administration alsoinclude pessaries, tampons, creams, gels, pastes, foams or spraycompositions containing such carriers as are known in the art to beappropriate.

Dosage forms for the topical or transdermal administration of acomposition of this invention include powders, sprays, ointments,pastes, creams, lotions, gels, solutions, patches and inhalants. Thecomposition may be mixed under sterile conditions with apharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to acomposition of this invention, excipients, such as animal and vegetablefats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays may contain, in addition to a composition of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays may additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a composition of the present invention to the body. Suchdosage forms may be made by dissolving or dispersing the composition inthe proper medium. Absorption enhancers may also be used to increase theflux of the composition across the skin. The rate of such flux may becontrolled by either providing a rate controlling membrane or dispersingthe composition in a polymer matrix or gel.

Ophthalmic compositions, eye ointments, powders, solutions and the like,are also within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise a cochleate of the invention in combination withone or more pharmaceutically acceptable sterile isotonic aqueous ornonaqueous solutions, dispersions, suspensions or emulsions, or sterilepowders which may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the composition isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity may be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the siRNA then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, and sodium chloride, inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating acomposition of the invention in the desired amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asnecessary, followed by filtered sterilization. Generally, dispersionsare prepared by incorporating the composition into a sterile vehiclewhich contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze-drying which yieldsa powder of the cochleate compositions of the invention plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease may be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectablecompositions are also prepared by entrapping the cochleates in liposomesor microemulsions which are compatible with body tissue.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, thecomposition can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the composition inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the composition.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compositions of the invention also can be prepared in the form ofsuppositories (e.g., with conventional suppository bases such as cocoabutter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the compositions of the invention are prepared withcarriers that will protect the composition against rapid eliminationfrom the body, such as a controlled release composition, includingimplants and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Methods for preparation of such compositions will beapparent to those skilled in the art. The materials can also be obtainedcommercially from Alza Corporation and Nova Pharmaceuticals, Inc.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of acomposition calculated to produce the desired therapeutic effect inassociation with the pharmaceutical carrier. The specification for thedosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the composition and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such a composition for the treatmentof individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration. Thepharmaceutical compositions can be included in a container along withone or more additional compounds or compositions and instructions foruse. For example, the invention also provides for packagedpharmaceutical products containing two agents, each of which exerts atherapeutic effect when administered to a subject in need thereof. Apharmaceutical composition may also comprise a third agent, or even moreagents yet, wherein the third (and fourth, etc.) agent can be anotheragent against the disorder, such as a cancer treatment (e.g., ananticancer drug and/or chemotherapy) or an HIV cocktail. In some cases,the individual agents may be packaged in separate containers for sale ordelivery to the consumer. The agents of the invention may be supplied ina solution with an appropriate solvent or in a solvent-free form (e.g.,lyophilized). Additional components may include acids, bases, bufferingagents, inorganic salts, solvents, antioxidants, preservatives, or metalchelators. The additional kit components are present as purecompositions, or as aqueous or organic solutions that incorporate one ormore additional kit components. Any or all of the kit componentsoptionally further comprise buffers.

The present invention also includes packaged pharmaceutical productscontaining a first agent in combination with (e.g., intermixed with) asecond agent. The invention also includes a pharmaceutical productcomprising a first agent packaged with instructions for using the firstagent in the presence of a second agent or instructions for use of thefirst agent in a method of the invention. The invention also includes apharmaceutical product comprising a second or additional agents packagedwith instructions for using the second or additional agents in thepresence of a first agent or instructions for use of the second oradditional agents in a method of the invention. Alternatively, thepackaged pharmaceutical product may contain at least one of the agentsand the product may be promoted for use with a second agent

EXEMPLIFICATION

The use of antisense oligonucleotides as therapeutic agents has beenwidely investigated in the past few years. Brantl, S. (2002) BiochimBiophys Acta. 1575 (1-3):15-25; Brent, L. J. N, et al. (2002) Neurosci.114(2):275-278; Akhtar et al. (1991) Nucleic Acids Res. 19: 5551. Theirefficacy is based on their ability to recognize their mRNA target in thecytoplasm and to block gene expression by binding and inactivatingselected RNA sequences. While the potential of antisense is widelyrecognized, limitations such as poor specificity, instability,unpredictable targeting and undesirable non-antisense effects hampertherapeutic use of antisense molecules. In addition, one of the majorlimiting aspects of this gene regulatory strategy is poor cellpenetration. Akhtar et al. (1991) Nucleic Acids Res. 19, 5551.

Intracellular delivery and concentration is necessary for antisenseinhibition of gene expression. It is believed that nakedoligonucleotides enter the cell via active processes of adsorptiveendocytosis and pinocytosis. However, naked antisense oligonucleotidesdo not appear to penetrate the endosomal barrier and gain access to thecytoplasmic compartment to any great extent. Lebedeva, I. et al. (2001)Annu. Rev. Pharmacol. Toxicol. 4:403-419; Weiss, B. et al. (1997)Neurochem. Int. 31:321-348). Although complexes of antisenseoligonucleotides with cationic liposomes have enhanced intracellulardelivery, they also have significant cytotoxicity. Their utility invitro and in vivo has also been limited by their lack of stability inserum and their inflammatory properties.

Compositions of the present invention utilize cochleates to achieveenhanced delivery of morpholino antisense molecules in vitro. Themorpholine backbone of these antisense molecules is not recognized bynucleases, and is therefore more stable. Morpholinos function by anRNase H-independent mechanism and are soluble in aqueous solutions, withmost being freely soluble at mM concentrations (typically 10 mg/ml toover 100 mg/ml). Nasevicius, A. et al. (2000) Nat. Genet. 26:216-220;Lewis, K. E. et al. (2001) Development 128:3485-3495; Mang'era, K. O. etal. (2001) Eur. J. Nucl. Med. 28:1682-1689; Satou, Y. et al. (2001)Genesis. 30:103-106; Tawk, M. et al. (2002) Genesis. 32:27-31. They arehighly effective with predictable targeting. Nasevicius, A. et al.(2000) Nat. Genet. 26:216-220; Lewis, K. E. et al. (2001) Development.128:3485-3495; Mang'era, K. O. et al. (2001) Eur. J. Nucl. Med.28:1682-1689; Satou, Y. et al. (2001) Genesis. 30:103-106; Tawk, M. etal. (2002) Genesis. 32:27-31.

Example 1 Preparation of Morpholino Cochleates

Rhodamine-labeled phosphatidyl ethanolamine (Rho-PE) liposomes wereprepared by adding dioleoylphosphatidylserine (DOPS) and Rho-PE at aratio of 20:1 (Rho-PE:DOPS) to chloroform at a ratio of 10 mg lipid/mlin a 50 ml sterile tube. The concentration of Rho-PE was approximately0.1% or 0.01% with respect to the DOPS.

The sample was blown down under nitrogen to form a film. Once dry, thesample was resuspended with TES buffer at a ratio of 10 mg lipid/ml. Theliposomes were then passed through a 0.22 μm filter. The homogenouspopulation of rhodamine-labeled liposomes were stored at 4° C. in theabsence of light under nitrogen.

Morpholinos were obtained from GeneTools, LLC (Philomath, Oreg.) for theGAPDH antisense sequence 5′ATCCGTTGACACCGACCTTCACCAT3′ (SEQ ID NO.: 1),and GAPDH mismatch sequence 5′ATCCCTTGAGACCGAGCTTCTCCAT3′ (SEQ ID NO.:2). These sequences have been used previously to target the first 25bases of the coding sequence and block GAPDH. They were solubilized byadding 0.834 ml TES [N-tris (hydroxymethyl)methyl-2-aminoethanesulfonicacid] buffer to the original bottle at neutral pH. The morpholino stockwas stored in 100 μl aliquots at −20° C. Prior to use, the aliquot mustbe heated at 65° C. for 5 min to ensure the morpholino hasn't droppedout of solution.

Approximately 400 μl of the fluorescent Rho-PE liposome suspension andabout 100 μl of the morpholino solution were added to a sterile glasstube and vortexed thoroughly (approximately 2 minutes). The samples werechecked for pH and observed both macro- and microscopically. The sampleswere observed to include liposomes and morpholino oligomers. The pH ofthe sample was slowly increased to approximately 8.0-8.5, by addition of1N NaOH. The sample was then vortexed for about 10 minutes. Thesuspension was then sonicated for about 2 minutes in a nitrogen gasatmosphere, and filtered using a 0.22 μm syringe filter. Morpholinos arehydrophilic non-charged molecules, and therefore do not interactstrongly with the liposomes. Raising the pH places a charge on the basepairs of the morpholino, favoring an interaction with the liposomes.

Cochleates were then formed by the slow addition (10 μl) of 0.1M calciumchloride to the suspension of Rho-PE liposomes and morpholino oligomersat a molar ratio of lipid to calcium of 2:1 with an external excess of 6mM calcium. The calcium chloride was added while vortexing using aneppendorf repeater pipette with a 500 microliter tip, adding 10 μlaliquots to the suspension every 10 seconds. At this point, the samplewas observed to include morpholino-cochleates. The sample was thenstored at 4° C. in the absence of light.

Samples were also prepared by lowering the pH to approximately 6.0-6.5as described above. As the pH was decreased from 7.4 to approximately6.0, an interaction was observed between the lipid and the morpholino,similar to that observed at pH 8.5.

Example 2 Delivery of Morpholinos Via Cochleates into Cells

Morpholino-cochleates were prepared as described in Example 1, withFITC-labeled GAPDH morpholinos. These morpholino-cochleates wereadministered to NGF differentiated rat P12 cells and photographed at 3hours and 12 hours as shown in FIGS. 1A and 1B, respectively, aftercochleate introduction. As illuminated by the fluoresced rhodamine usingLCSM fluorescence imaging, the cochleates fuse with the outer membraneand form submembrane aggregates. FIGS. 1C (low power) and 1D (highpower) are photographs of flouresced rhodamine labeled cochleatescontaining fluorescein isothiocyanate (FITC) labeled morpholinos. FIGS.1C and 1D depict cochleates containing morpholinos, morpholinos thathave been released into the cytosol from unwrapped cochleates, and thedelivery of FITC labeled anti-GAPDH Morpholino into the cytoplasm. Themorpholinos delivered into the cells depicted in these FIGS. 1A-D wereretained in the cells for at least 72 hours. The labeled morpholinoswere delivered into the cell cytosol and nucleus (FIGS. 1C and 1D).

As shown in FIGS. 4A-B, the cochleates fuse or are taken up by the cellsand form submembrane aggregates. FIG. 4A shows intracellular rhodaminecochleates (punctuate and diffuse red color) after 3 hours. Yellow,yellow-green and orange-red indicate cochleates containing morpholinos.FIG. 4B shows delivery 12 hours after cochleate introduction. Rhodaminelabeled lipid, originally in cochleates, is largely distributed to thecellular membranes although there appears to be accumulation within thecell, suggesting that some empty cochleates may be sequestered invacuoles, while FITC-labeled morpholinos (green color) have beenreleased into the cytosol from unwrapped cochleates by 12 hr. afterinitial presentation to the cell.

As shown in FIG. 3, Western blotting for GAPDH protein in these labeledcells showed a time dependent decrease in GAPDH protein levels by 18-24hours following treatment with cochleates with GAPDH antisensemorpholino (lower blot) while control cells receiving vehicle withcochleate alone (upper blot) showed no change in GAPDH protein levels.This example demonstrates that morpholino-cochleates are an efficienttechnique for delivering antisense morpholinos in a manner that does notcompromise the integrity of the cells. Plain cochleates or cochleateswith sense morpholino at the same concentration had no toxicity.

Example 3 Delivery of Morpholinos Via Cochleates Into Retinal GanglionCells

Morpholino-cochleates were prepared as described in Example 1 withFITC-labeled GAPDH morpholinos. These morpholino-cochleates wereadministered to retinal ganglion cells in situ in retinal organotypeculture. It was observed that the morpholino-cochleates readilyinteracted with the cells in the retinal ganglion cell layer (FIGS. 2Aand 2B). FIGS. 2A and 2B are images of X-Y RGCL LCSM computationalslices demonstrating avid cochleate uptake by retinal ganglion cells insitu. Scale bars indicate 10 micrometers.

FIG. 2A indicates cochleate delivery and biological activity of theantisense molecules. Interference with GAPDH by the antisense moleculetriggers apoptosis, detected here by YOYO staining of all the retinalganglion cells in the field. Cell nuclei with apoptotic chromatincondensation have very bright homogeneous YOYO signals (See FIG. 2B).This system provides a very efficient technique for delivering antisenseoligonucleotides in a manner that does not compromise the integrity ofthe cells. In sharp contrast, other delivery methods were associatedwith some cytotoxity and a maximum of 10% transfection.

Example 4 Cochleate Delivery of Antisense DNA in a Murine Model ofChronic Lymphocytic Leukemia

NZB mice develop a B-1 cell lymphoproliferative disorder that serves asa murine model of chronic lymphocytic leukemia (CLL). These malignantB-1 cells produce significantly higher levels of IL-10 mRNA than normalB-1 or B cells. The addition of antisense oligodeoxynucleotides specificfor IL-10 mRNA dramatically inhibits the growth of leukemic B-1 cells ina time and dose dependent manner. Control cell lines that do not dependon IL-10 for growth are not affected. In vitro antisense therapytargeted at the 5′ region of the IL-10 mRNA not only resulted ininhibition of malignant B-1 cell proliferation, but also inhibited IL-10production by malignant B-1 cells. In vivo, antisense therapy waseffective in preventing death of the animals due to uncontrolled growthof 5×10⁶ intraperitoneally transferred malignant B-1 cells. In theseexperiments, after approximately 6 weeks, at which time the controlanimals had all died, the antisense IL-10 treated groups had no evidenceof disease.

Comparison of Cochleates and Mini-Osmotic Pump

Cochleates were much more effective in delivering antisense IL-10oligonucleotide for preventing the growth of malignant B-1 cells invivo, and protecting against disease and death. Using a mini-osmoticinfusion pump, 300 ug/day phosphorothioate-modified antisense IL-10oligonucleotide was constantly infused for 28 days, totaling 8.4 mg.Lower quantities or shorter times did not result in protection fromtumor cell challenge.

Although phosphorothioate oligos are known to have much longer halflives in vivo, much lower doses of unmodified phosphodiester antisenseIL-10 oligonucleotides were effective when formulated and delivered incochleates. Four injections (days 0, 5, 8, and 14) of antisense IL-10cochleates, totaling 1.3 mg, prevented the growth of malignant B-1cells. The reasons for this increased efficacy of unmodified antisenseIL-10 when delivered in cochleates may be due to the prevention ofnuclease degradation in plasma and interstitial fluid, delivery of theintact oligonucleotide directly into the cytoplasm of the target cell,and/or slow delivery over a prolonged period of time, due to themultilayered nature of the cochleates.

Cochleate Delivery of Antisense DNA Protects Against B Cell Lymphoma

FIG. 5 summarizes in vivo antisense IL-10 experiments including bothforms of delivery, pumps and cochleates. All mice were (NZB DBA/2) F1recipients that received a transfer of leukemic B-1 cells. The percentof diseased animals in a particular treatment group is the number ofdiseased animals divided by the animals studied, multiplied by 100.Disease includes all mice that died before day 60 with hind legparalysis or evidence of clones of malignant B-1 cells detected by flowcytometry or abnormal pathology at the time of sacrifice. Mini infusionpump antisense IL-10 treated animals (0/4), Antisense IL-10-Cochleatetreated animals (0/3), sense IL-10 (4/5), control (receiving eitherpumps and buffer alone or cochleates alone) (7/7), untreated receivingno treatment following transfer of the malignant B-1 cells (7/7).

This example demonstrates successful cochleate delivery of an antisensemolecule in vivo, wherein biological activity was retained.

Example 5 Delivery of Morpholino-Cochleates to Rats with InducedParkinson's Disease-Like Pathology

Parkinson's disease-like pathology with be induced in rats withCSF-delivered rotenone and joint rotenone-CLβL cerebrospinal fluid (CSF)delivery. Morpholino-cochleates will be used to deliver morpholinos tosuppress GAPDH and p53 protein levels to study its effect on NSdnapoptosis and protein aggregation caused by the CSF infusion.

Based on studies in cultured cells and the findings of Greenamyre etal., it is expected that chronic CSF-delivered rotenone will inducePD-like pathology in the rats at lower dosages than those found for ratswith intravenous delivery and that joint rotenone-CLβL CSF delivery willmarkedly shift the concentration dependence to lower values. Based onprevious studies in culture, it is expected that both the p53 and GAPDHantisense will reduce NSdn apoptosis and may also decrease any proteinaggregation caused by the CSF infusion. The GAPDH antisense treatmentshould not affect p53 levels or subcellular localization while the p53antisense treatment should prevent both GAPDH upregulation and nuclearaccumulation.

Morpholino-cochleates will be employed to induce non-lethal reductionsin GAPDH (see FIG. 3 in which GAPDH was reduced to about 50%), and p53proteins. High GAPDH morpholino-cochleate concentrations resulting inmarked GAPDH reductions cause cellular death over 3 to 8 hours, probablydue to a failure of glycolysis. The morpholino oligos from Example 1from GeneTools, LLC (Philomath, Oreg.) will be used. For p53 antisenseand mismatch, 5′TCATATCCGACTGTGAATCCTCCAT3′ (SEQ ID NO.: 3) and5′TCATTTCCGTCTGTGTATCCTGCAT3′ (SEQ ID NO.: 4), respectively, will beused. These sequences have been used to block GAPDH and p53 synthesis(Chen et al. (1999) J Neurosci. 19:9654-62; Fukuhara et al. (2001)Neuroreport 12:2049-52), and target the first 25 bases of each codingsequence. Three other series of sequences will be used that havepreviously altered either p53 or GAPDH synthesis.

Since both the cochleates and the morpholinos can be fluorescentlylabeled as described above and are retained in cells afterparaformaldehyde fixation, it should be possible to observe theproportion of cells that concentrate the carrier and the morpholinobefore collecting lysates from ventral mesencephalon to measure theoverall reduction in GAPDH or p53 protein. Furthermore, fluorescencelabeling should allow a determination of whether specific SNc cellularphenotypes showing evidence of apoptosis or protein aggregation alsotook up the cochleates.

Similar to preliminary studies with retrograde tracers (Yee et al.,(1994) Cell Mol Neurobiol 14:475-86; Shimizu et al. (2001) J Cereb BloodFlow Metab 21:233-43), the antisense and mismatch oligonucleotides willbe infused into the lateral ventricle using a cannulae system.Morpholinos will be carried into NSdns and other cells from the CSF bythe cochleates. Zarif et al. (2000) Adv Exp Med Biol 465:83-93.Rhodamine can be included in the lipid constituents of the cochleatesand will allow the visualization of the binding of the cochleate to theouter membranes of the cell using LCSM fluorescence imaging asillustrated in FIGS. 1A-D.

Both the oligomeric specific and oligomeric non-specific antibodies willbe employed to determine whether all or part of the immunoreaction in aspecific subcellular locus is due to a specific oligomer of GAPDH.Monoclonal antibody that specifically recognizes GAPDH monomer or dimerbut not GAPDH tetramer will be obtained from Ono Pharmaceuticals(Japan). Also used will be a sheep polyclonal antibody that onlyrecognizes GAPDH tetramer and a mouse monoclonal antibody thatrecognizes all oligomeric forms of GAPDH. Carlile et al. (2000) Mol.Pharmacol. 57:2-12. Co-staining with YOYO-1 will be used todifferentiate the nuclear and non-nuclear compartments. Carlile et al.(2000) Mol. Pharmacol. 57:2-12.

In order to quantitate immunofluorescence for different antibodies anddifferent treatments, sections will be incubated for differenttreatments together to ensure identical exposure to antibodies.Fluorescence intensity will be measured from within 3 extra nuclear 4μm×4 μm regions within the somata of randomly chosen neurons using theprogram Northern Eclipse (Empix Imaging, Mississauga, Ontario). Threemeasurements will be made immediately outside each neuronal somata inorder to determine intraneuronal fluorescence above background. Tsuda etal. (1994) Neuron 13:727-36. Approximately 600 neurons will be examinedfor each animal and 7200 for each concentration time. The program allowsthe coordinates of each measurement to be retained so that measurementscan be made for identical loci from simultaneously collected images fordifferent antibodies.

It is expected that both p53 tumor suppressor protein and GAPDH willundergo increases and nuclear accumulation in response to rotenone orCLβL exposure. Rotenone will only increase the proteins in NSdns whileCLβL will increase them in all SNc cells. It is expected that aproportion of the neurons will show dense nuclear immunofluorescence forthe antibody against GAPDH monomer/dimer and for the antibody againstall GAPDH oligomeric forms, but not for the antibody that onlyrecognizes tetramer. A follow-up study has been conducted on our studiesin Parkinson's Disease (PD) postmortem SNc (Tatton, Exp Neurol 166:29-43(2000)), using the antibody that recognizes all GAPDH oligomers withsimilar examinations using the monomer/dimer selective antibody. It wasfound that GAPDH nuclear accumulation in PD postmortem SNc involves onlythe monomer/dimer. It will be valuable to determine if the model showsthe same oligomeric selectivity.

Example 6 siRNA-Cochleates for the Treatment of Fungal Infections

These studies will determine the relative effectiveness ofsiRNA-cochleate compositions for preventing invasive Aspergillosis inanimal models that mimic disease in humans. Female BalbC or DBA2 micefrom Charles River Labs will be used for this study because they behavein a reliable manner when infected with fungal pathogens. Previousstudies have shown that intravenous inoculation with pathogenic fungi inmice produces an infection similar to that seen in man.

Aspergillus fumigatus H⁺-ATPase will be studied as an effect therapeutictarget for antifungal agents employing siRNA-cochleates of theinvention. The plasma membrane H⁺-ATPase from Candida albicans wascloned and characterized. Monk, B. C., et al. (1991) J Bacteriol.173(21): 6826-36. Similar cloning and characterization projects havebeen completed on plasma membrane H⁺-ATPases from Cryptococcusneoformans (Soteropoulos, P., et al. (2000) Antimicrob Agents Chemother44(9): 2349-55) and Aspergillus fumigatus (Burghoom, H. P et al. (2002)Antimicrob Agents Chemother 46(3):615-24).

The gene, AfPMA1, encoding the plasma membrane proton pump (H⁺-ATPase)of Aspergillus fumigatus was characterized from Aspergillus fumigatusstrain NIH 5233 and clinical isolate H11-20. An open reading frame of3109 nucleotides with two introns near the N-terminus predicts a proteinconsisting of 989 amino acids with a molecular weight of approximately108 kDa. The predicted Aspergillus fumigatus enzyme is 89% and 51%identical to H⁺-ATPases of A. nidulans and S. cerevisiae, respectively.AfPMA1 is a typical member of the class III P-type ATPase family thatcontains 10 predicted transmembrane segments and conserved sequencemotifs, TGESL (SEQ ID NO.: 13), CSDKTG (SEQ ID NO.: 14), MXTGD (SEQ IDNO.: 15) and GDGXNDXP (SEQ ID NO.: 16) within the catalytic region. Theenzyme represents 2% of the total plasma membrane protein, and it ischaracteristically inhibited by orthovanadate with an IC₅₀˜0.8 μM. TheH⁺-ATPase from Aspergillus spp. contains a highly acidic insertionregion of 60 amino acids between transmembrane segments 2 and 3 whichwas confirmed in the membrane assembled-enzyme with a peptide-derivedantibody. Increasing gene copy number of AfPMA1 confers enhanced growthin low pH medium consistent with its role as a proton pump. Burghoom, H.P. et al. (2002) 46(3):615-24.

Cell Phenotype and Morphology Changes will be Evaluated for Aspergillusfumigatus

The normal septate hyphae are wide and form dichotomous branching, i.e.,a single hypha branches into two even hyphae, and then the myceliumcontinues branching in this fashion. It was observed that sublethalamount of anti-H+-ATPase antagonists like ebselen produce long thinhyphal elements with diminished branching. As the H⁺-ATPase activity isdiminished, increasing cell surface area helps maintain the overallcapacity of the system by increasing the number of pumps. It isexpected, that stressing the mutant proton pumps by acidifying thecytoplasm with weak acids at low external medium pH will show similarresults. Aspergillus is particularly resistant to high temperature andgrows efficiently at 45° C. It was observed that the MIC for cellkilling with ebselen is decreased with increasing temperature. Whetherinhibition of PMA1 alters the temperature profile for growth will bedetermined. Finally, spore formation and spore germination will beexamined in a similar manner.

The essential role of the H+-ATPase in spore germination andmultiplication of growing cells provides an opportunity to explore theability of nanocochleates to efficiently deliver siRNAs targeted to theH+-ATPase of A. fumigatus. Given the medical importance of A. fumigatusand the paucity of available antifungal compounds, siRNA cochleatecompositions have the potential to be effective therapeuticalternatives.

The goal is to determine the feasibility and technical merit ofpreparing and testing stabilized siRNA-cochleate compositions that willenhance the antifungal activity of these oligonucleotides.

Stable compositions of nanocochleates containing siRNA targetingAspergillus fumigatus H⁺-ATPase are to be prepared, and that thesecompositions will be capable of interacting with and inhibiting thegrowth of Aspergillus fumigatus both in vitro and in vivo.

Standard protocols will be used to prepare siRNA-cochleates. Lipid (PS)to siRNA ratios will range from 25:1 to 100:1, wt:wt. Purified siRNAmolecules will be purchased from commercial vendors.

To stabilize the particle size of the cochleate compositions, severalcommercially available, FDA approved excipients will be evaluated fortheir ability to stabilize size characteristics of the cochleatecompositions. Excipient candidates will be chosen that have thepotential to interact with the cochleate surface and preventcochleate-cochleate interaction. E.g., excipients that containhydrophilic polymers (e.g., polyethyleneglycol (PEG)) with/or without ahydrophobic tail can be used. Excipients with properties that mimiccasein, which is a highly phosphorylated, calcium-binding molecule alsowill be tested.

Aspergillus fumigatus PMA1 siRNA

To investigate gene silencing by RNA interference as a potentialtherapeutic in pathogenic fungi, small interference RNA (siRNA) weredesigned to the PMA1 gene of Aspergillus fumigatus. PMA1 encodes theessential plasma membrane proton-ATPase, which regulates electrochemicalproton gradients and intracellular pH in this pathogenic organism.Miller, M. D. et al. (1992) J Exp Med 176:1739-1744. Two PMA1 sense andantisense siRNA pairs (21 mers) were designed to regions 100 and 162nucleotides downstream of the start codon (Genbank/NCBI accession numberAY040609) using Qiagen's siRNA design tool(http://python.penguindreams.net/Xeragon_Order_Entry/jsp/SearchByAccessionNumber.jsp).The siRNA sequences are listed in Table I. Burghoorn, H. P. et al.(2002) Antimicrob Agents Chemother 46:615-24. TABLE I A. fumigatus PMA1siRNA sequences cDNA Target Sense siRNA Antisense siRNA Sequence Region(5′-3′) (3′-5′) AACCGTTACATC 100 nt downstream CCGUUACAUCUCdTdTGGCAAUGUA TCGACTGCT from start codon GACUGCUdTdT GAGCUGACGA (SEQ IDNO.: 5) (SEQ ID NO.: 6) (SEQ ID NO.: 7) AAGCCTCCAGCA 162 nt downstreamGCCUCCAGCAGA dTdTCGGAGGUCG GAAGAAGAA from start codon AGAAGAAdTdTUCUUCUUCUU (SEQ ID NO.: 8) (SEQ ID NO.: 9) (SEQ ID NO.: 10)

The characterization of siRNA cochleate compositions will be performedusing standard protocols including biochemical analysis (lipid and drugquantitation and integrity), morphology (light and electron microscopy),and particle sizing.

Imaging, General Methods.

An Olympus FV500 confocal laser scanning microscope will be used toobtain images of fluoresce-tagged cochleates in real time from livingcells and from fixed tissue in order to investigate the dynamics ofcochleate uptake by macrophages and fungus. A Hitachi S4700 fieldemission scanning electron microscope (FESEM) will be used toinvestigate details of cochleate ultrastructure and provide ultra-highresolution images of cochleate-cell membrane/wall interactions.

Confocal Imaging

A) Cochleate uptake over time. Cells/fungi will be exposed to cochleatesfor 2, 5, 10, 15, 30, 60 min., 12 h, 24 h, 48 h to establish a temporalsequence for uptake and dispersal. Cells/fungi will be fixed with 2%gluteraldehyde (EM grade) and will be exposed to other cell markers forcolocalization. For real-time imaging, coverslips are transferred to aenvironmentally controlled sealed chamber.

B) Subcellular localization. Cell nuclei marked with Alexa 488-histone 1will be used in live cells to determine whether siRNA localizes in thenucleus. Lysotracker probes, which mark lysosomes, will help determineif cochleates and/or their contents are found in lysosomes. Tarasova N.I. et al. (1997) J. Biol. Chem. 272: 14817-14824. A cationic linearpolyene, TMA-DPH, a lipid marker for endocyotsis and exocytosis,(Kawasaki Y. et al. (1991) BBBA 1067: 71-80; Illinger D. et al. (1993)Biol. Cell 79: 265-268) will assist in determining how cochleates areabsorbed/released by macrophages. All markers are available at MolecularProbes (Oregon)

FESEM Imaging

Rapid freezing of unfixed, bulk biological samples such as cochleatesand cells/fungi will produce the least distortion in the specimens.However, it will be necessary to test alternate protocols to see whatadditional information about cochleate/cell interactions may beobtained.

A) Double Coating of Unfixed, Frozen, Hydrated Samples. A nitrogen slushwill be used to cool down unfixed, hydrated biological samples and thenperform a double coating layer (2 nm platinum, followed by 5-10 nMcarbon) to allow high resolution backscattered electron detection(Walther P. et al. (1997) Scanning 19:343-348).

B) Cryopreserved Samples. 30% sucrose and 20% sucrose/3% PEG-400cryopreservation methods will be tested on fixed samples. This methodwill likely not provide ultra high resolution images as indouble-coating, but may be best for preparing composite and topographicEM images at lower accelerating voltages.

C) Fixed Dehydrated, Critical Point Dried Samples. Method A may be toodamaging to fungi, in which case a critical point drying method (Muller,W. H. et al. (2000) 22:295-303) will be used that can also be used formammalian cells to provide high resolution imaging. This method willprovide high resolution images. However, this method can produce moredistortion of the sample than A or B.

Example 7 Methods of Making siRNA-Cochleates Directed Against theExpression of erbB Protein

Labelled siRNA (Sense: 5′-UCCCGAGGGCCGGUAUACATT-3′ (SEQ ID NO.: 11);Antisense: 5′-UGUAUACCGGCCCUCGGGATT-3′ (SEQ ID NO.: 12) directed againsterbB (targeting codons 852-873 of the mRNA encoding erb) were obtainedfrom PPD, Inc. (Wilmington, N.C.). siRNA were Cy5 labelled or FITClabeled for various experiments as indicated in the below experiments.The annealed 22 bp siRNA 20 micromolar stock solution included 0.26 μgsiRNA/μl. The siRNA buffer contained either (i) 100 mM potassiumacetate, 30 mM HEPES-KOH (pH7.4), and 2 mM magnesium acetate; or (ii) 20mM KCl, 6 mM HEPES (pH 7.5), and 0.2 mM magnesium chloride.

Stock liposome suspensions were prepared by solubilizing DOPS powderalone, or DOPS powder (99% by weight) and Rhodamine PE (1% by weight),in chloroform, drying to film under nitrogen, and rehydration in TESbuffer (pH 7.4) to a concentration of 10 mg/ml by vortexing.

Cochleates were prepared from liposomes as described above using thefollowing methods. In the following methods, unless otherwise indicated,the DOPS:siRNA weight ratio was 50:1, and methods were carried out atneutral pH conditions with approximately isotonic salt concentrations.

Trapping Method. 6.5 μl of stock liposome suspension was filtered with a0.2 micron filtration membrane to obtain small unilamellar vesicleliposomes (SUVs). 5 μl of the stock siRNA solution (1.3 μg siRNA) wasplaced in an Eppendorf micro-centrifuge tube, and the 6.5 μl SUVsuspension (65 μg DOPS lipid) was added to the siRNA. 88.5 μl TES Bufferwas added and mixed well, followed by addition of 8 μl of 0.1M calciumchloride and mixed well to form cochleates.

Extrusion Method. 5 μl of the 20 μmM stock siRNA solution (1.3 μg siRNA)was placed in an Eppendorf micro-centrifuge tube. 6.5 μl DOPS liposomesuspension (65 μg lipid) from the 10 mg/ml suspension was added to thesiRNA. 88.5 μl of TES buffer was added and the mixture extruded 7 timeswith an Avanti mini-extruder, which allows production of unilamellarlipid vesicles by multiple extrusions between two connected syringesthrough a polycarbonate membrane with defined pore size. Membrane usedwas 0.2 microns pore size. 7 μl of 0.04M calcium chloride was added andmixed well to form cochleates.

Alternative Extrusion Method 25 μl of the 20 μmM stock siRNA solution(6.5 μg siRNA) was placed in an Eppendorf micro-centrifuge tube. 25 μlunfiltered DOPS liposome suspension (250 μg lipid) from the 10 mg/mlsuspension was added to the siRNA. The mixture was extruded 7 times. 6μl of 0.05M calcium chloride solution was added and mixed well to formcochleates. The lipid:siRNA ratio was 39:1 wt/wt.

Cochleate Conversion Method. 65 μl DOPS liposome suspension from the 10mg/ml suspension was placed in an Eppendorf micro-centrifuge tube. 4 μlof 0.1M calcium chloride solution was added and mixed well to formcochleates in suspension. 7 μl of this suspension was added to anothermicro-centrifuge tube, and centrifuged at 13.00 RPM for 30 minutes. Thesupernatant was removed, and 5 μl of the 20 μmM stock siRNA solution(1.3 μg siRNA) was added to the cochleates. 3 μl of 150 mM EDTA wasadded to convert the cochleates to liposomes associated with the siRNA.8 μl of 0.01M calcium chloride solution was added and mixed well,followed by the addition of 80 μl of TES buffer and 2 mM calciumchloride and mixing.

Example 8 siRNA-Cochleates Directed Against the Expression of erbBProtein

Employing the siRNA identified and liposome stock solutions prepared inExample 7, siRNA cochleates were prepared and administered to ovariancancer cell line SKOV3 (PPD, Inc.). In the following methods, theDOPS:siRNA weight ratio was 2:1 (12.45 μg lipid, 6.5 μg siRNA). Thelipid concentration in the liposomes was about 100 μg/ml (0.1 mg/ml),the siRNA concentration approximately 57 μg/ml (0.057 mg/ml). SKOV3cells were grown in monolayers in humidified air with 5% CO₂ at 37° C.in 60 mm Petri dishes (Corning) containing 5 mL of DMEM supplementedwith 10% FBS. The calcium concentration in mix was 130 mM for theelevated or high calcium method, and 3.8 mM for the low or depressedcalcium method.

High Calcium Method. 25 μl of the 20 μM stock siRNA solution was placedin an Eppendorf micro-centrifuge tube to which 6 μl of 2.5M calciumchloride solution was added and mixed well. 83 μl of DOPS liposomes at150 μg/ml in TES (pH 7.0) was added, followed by 17 μl of TES Buffer.The total volume (131 μl) was mixed well. 5 μl of the mixture was thenadded to 45 μl cell culture medium and incubated for 72 hours at 37° C.The culture was fixed and stained with antibodies for erbB2 expression.

Low Calcium Method. 25 μl of the 20 μmM stock siRNA solution was placedin an Eppendorf micro-centrifuge tube to which 5 μl of 0.1M calciumchloride solution was added and mixed well. 83 μl of DOPS liposomes at150 μg/ml in TES (pH 7.0) was added, followed by 17 μl of TES Buffer.The total volume (131 μl) was mixed well. 5 μl of the mixture was thenadded to 45 μl cell culture medium and incubated for 72 hours at 37° C.The culture was fixed and stained with antibodies for erbB2 expression.

Control Preparations. SKOV3 cells were also incubated with emptycochleates made employing the same methods (except without addition ofsiRNA) and Lipofectamine formulated siRNA. The cultures were also fixedand stained with antibodies for erbB2 expression.

Partial Knockdown of erbB2 in SKOV3 Cells

Absorption results (ELISA assay) for surface erb B2 expression for eachof the treated cultures are shown in FIG. 6. Decreased absorptionindicates inhibition of Erb B2 by specific or non-specific mechanisms.The low calcium siRNA cochleate composition (LCaRNAcc) did not appear toinhibit Erb B2 compared to empty cochleates (LCaEPTcc). Whereas, thehigh calcium siRNA-cochleate composition did (HCaRNAcc vs. HCaEPTcc).Less staining of wells treated with Lipofectamine formulated siRNA(lipo-RNAamb and lipo-RNA-Cy5) may be due to specific inhibitioncombined with non-specific down regulation and fewer cells associatedwith greater cellular cytotoxicity.

FIG. 7 is a series of fluorescent confocal microscopy images of theSKOV3 cells 24 hours post-exposure to: empty cochleates (panel A), 1%rhodamine-labelled cochleates (panel B), anti-erb B2 siRNA-cochleates(panel C), and Cy5 labelled anti-erb B2 siRNA-cochleates (panel D). Thecochleates images in panel C and panel D were manufactured using thehigh calcium method described above. Partial knockdown of cytoplasmicerb B2 by anti erb B2 siRNA-cochleates was observed (panel C and panelD), as well as confirmation of intracellular delivery and localizationof cochleates and siRNA around the nucleus (panel B and panel D).Additionally, the subcellular distribution of Rhodamine-labelledcochleates (panel B) and Cy5 siRNA-cochleates (panel D) appears to bedifferent, indicating delivery and release of siRNA.

FIG. 8 is a series of confocal microscopy images of SKOV3 cells 24 hourspost-exposure to: empty cochleates (panel A), 1% rhodamine-labelledcochleates (panel B), anti-erb B2 siRNA-cochleates (panel C), and Cy5labelled anti-erb B2 siRNA-cochleates (panel D). Partial knockdown ofmembrane-localized erbB2 in SKOV3 cells after exposure to siRNA(erbB2)cochleates was observed (panel C and panel D). Also, intracellulardelivery of rhodamine cochleates (panel B) and Cy5 labeled siRNA (panelD) is observed.

Example 9 Cochleates Prepared with siRNA-PEI Complexes

siRNA and polyethylenimine (PEI) were allowed to associate to form apositively charged complex and then bound to negatively chargedliposomes and encochleated. The effect of these encochleated complexeswas studied.

22.5 μl of siRNA (20 μM) was added to an Eppendorf micro-centrifugetube. 16.2 μl of PEI (2000 MW, Lupasol G35, BASF) at a concentration of0.05%, was added and mixed well. Then, 116 μl of pre-made DOPS liposomeat 1.5 mg/ml (in TES, pH7.0) was added to this mixture and mixed well.Finally, 115 μl of 0.1M calcium chloride was added and mixed well toform cochleates. Cochleate morphology was confirmed microscopically. Inorder remove any free (unencochleated) siRNA from the siRNA-cochleatecomposition, the siRNA-cochleates were pelleted by centrifugation andthe supernatants removed. Pellets were re-suspended.

Cochleates also were formed with non-specific siRNA (no specificityagainst erbB2 and no known intracellular target) according to the samemethod. The anti Erb siRNA-cochleates and non-specific siRNA-cochleates(both formed with PEI) were administered to SKOV3 cells at 0.25 μg (fulldose) and 0.125 μg (50% dose), alongside untreated SKOV3 cells and wereincubated for 72 hours.

As summarized in FIG. 9, SKOV3 cells treated with thesiRNA/PEI-cochleate compositions (Erb_siRNA/PEI/Cch.Plt (1)), showed asignificant reduction in Erb B staining compared to untreated cells(Cell only (1)). Analogous compositions with a non-specific siRNA showedstatistically less inhibition (CtrlErb_siRNA/PEI/Cch.Plt (1)). When halfthe concentration of siRNA/PEI-cochleates were used, the anti-Erb BsiRNA/PEI-cochleates (ErbB Plt(2)) continued to cause a significantreduction in Erb B staining, but the control cochleates (Ctl.Plt(2))showed no inhibition compared to untreated cells (Cell Only (2)). Thisindicates an anti-ErbB2-specific effect of the cochleate deliveredsiRNA.

The siRNA/PEI-cochleates were compared to SKOV3 cells treated with (1)unencochleated siRNA/PEI complex, (2) encochleated Fetal Bovine Serum(FBS) and PEI, (3) unencochleated FBS and PEI, and untreated cells.These controls were formulated by the same methods and in the samequantities and concentrations as the siRNA cochleates.

As summarized in FIG. 10, greater inhibition of Erb B was seen uponadministration of siRNA/PEI-cochleates (Erb_siRNA/PEI/Cch.Plt(1)), ascompared to the unencochleated siRNA/PEI (Erb_siRNA/PEI/Cplz.Plt(1)),indicating a positive role for cochleate delivery of siRNA.FBS/PEI-cochleates (FBS/PEI/Cch.Plt(1)), and unencochleated FBS/PEI(FBS/PEI/CplxPlt(1)), showed a decrease in staining due to cytotoxicityof un-complexed PEI.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An siRNA-cochleate composition comprising: a cochleate; and an siRNAassociated with the cochleate.
 2. The siRNA-cochleate composition ofclaim 1, wherein the siRNA comprises at least one mismatch.
 3. ThesiRNA-cochleate composition of claim 1, wherein the siRNA comprises atleast one substitution.
 4. The siRNA-cochleate composition of claim 1,wherein the siRNA is about 21-23 nucleotides long.
 5. ThesiRNA-cochleate composition of claim 1, wherein the siRNA mediates RNAinterference against a target mRNA.
 6. The siRNA-cochleate compositionof claim 5, wherein the target mRNA expresses a protein selected fromthe group consisting of: a cancer protein, a virus protein, an HIVprotein, a fungus protein, a bacterial protein, an abnormal cellularprotein, a normal cellular protein.
 7. The siRNA-cochleate compositionof claim 1, further comprising a second siRNA directed against a secondtarget mRNA.
 8. The siRNA-cochleate composition of claim 1, wherein thecochleate comprises a negatively charged lipid component and amultivalent cation component.
 9. The siRNA-cochleate composition ofclaim 1, wherein the siRNA is complexed with a transfection agent priorto contacting the liposomes.
 10. The siRNA-cochleate composition ofclaim 9, wherein the transfection agent is a polycationic transfectionagent.
 11. The siRNA-cochleate composition of claim 9, wherein thetransfection agent is polyethylenimine (PEI) or a derivative thereof.12. The siRNA-cochleate composition of claim 1, further comprising atleast one additional cargo moiety.
 13. The siRNA-cochleate compositionof claim 1, further comprising an aggregation inhibitor.
 14. A method ofadministering an siRNA to a host comprising: administering abiologically effective amount of an siRNA-cochleate composition to ahost comprising a cochleate and an siRNA associated with the cochleate.15. The method of claim 14, wherein the siRNA is delivered from thecochleate to a cell in the host.
 16. The method of claim 15, wherein thesiRNA is delivered into a cytosol compartment of the cell.
 17. Themethod of claim 14, wherein the siRNA mediates RNA interference againsta target mRNA in the host.
 18. The method of claim 14, wherein targetmRNA expression in the host is reduced by at least about 50%.
 19. Themethod of claim 11, wherein target protein synthesis in the host isreduced by at least about 10%.
 20. The method of claim 11, whereintarget protein synthesis in the host is reduced by at least about 50%.21. The method of claim 11, wherein the host is a cell, a cell culture,an organ, tissue, or an animal.
 22. The method of claim 11, comprisingthe step of examining the function of the target mRNA or proteinexpressed by the target mRNA in the host.
 23. A method of treating asubject having a disease or disorder associated with expression of atarget mRNA, comprising: administering to a subject a therapeuticallyeffective amount of an siRNA-cochleate composition, comprising acochleate and an siRNA against a target mRNA associated with a diseaseor disorder, such that the disease or disorder is treated.
 24. Themethod of claim 23, wherein the disease or disorder is selected from thegroup consisting of: a neurological disorder associated with aberrant orunwanted gene expression, schizophrenia, obsessive compulsive disorder(OCD), depression, a bipolar disorder, Alzheimer's disease, Parkinson'sdisease, a lysosomal storage disease, Fabry's disease, Gaucher'sDisease, Type I Gaucher's Disease, Farber's disease, Niemann-Pickdisease (types A and B), globoid cell leukodystrophy (Krabbe's disease),metachromic leukodystrophy, multiple sulfatase deficiency, sulfatidaseactivator (sap-B) deficiency, sap-C deficiency, G_(M1)-gangliosidosis,Tay-Sachs disease, Tay-Sachs B1 variant, Tay-Sachs AB variant, AcidMaltase Deficiency, Mucopolysaccharidosis, Sandhoff's disease, a cancer,a cell proliferative disorder, a blood coagulation disorder,Dysfibrinogenaemia, hemophelia (A and B), dematological disorders,hyperlipidemia, hyperglycemia, hypercholesterolemia, obesity, acute andchronic leukemias and lymphomas, sarcomas, adenomas, a fungal infection,a bacterial infection, a viral infection, an autoimmune disorder,systemic lupus erythematosis, multiple sclerosis, myasthenia gravis,autoimmune hemolytic anemia, autoimmune thrombocytopenia, Grave'sdisease, allogenic transplant rejection, rheumatoid arthritis,ankylosing spondylitis, psoriasis, scleroderma, carcinomas, epithelialcancers, small cell lung cancer, non-small cell lung cancer, prostatecancer, breast cancer, pancreatic cancer, hepatocellular carcinoma,renal cell carcinoma, biliary cancer, colorectal cancer, ovarian cancer,uterine cancer, melanoma, cervical cancer, testicular cancer, esophagealcancer, gastric cancer, mesothelioma, glioma, glioblastoma, pituitaryadenomas, inflammatory diseases, osteoarthritis, atherosclerosis,inflammatory bowel diseases (Crohns and ulcerative colitis), uveitis,eczema, chronic rhinosinusitis, asthma, a hereditary disease, cysticfibrosis, and muscular dystrophy.
 25. A method of forming ansiRNA-cochleate composition comprising: precipitating a liposome and ansiRNA to form an siRNA-cochleate.
 26. The method of claim 25, comprisingadjusting the pH of the siRNA.
 27. The method of claim 25, comprisingcharging the base pairs of the siRNA.
 28. The method of claim 25,wherein the siRNA is complexed with a transfection agent prior toprecipitating.
 29. The method of claim 28, wherein the transfectionagent is mixed with the liposomes prior to adding the siRNA.
 30. Themethod of claim 28, wherein the transfection agent is PEI or aderivative thereof or other polyvalent cation.
 31. The method of claim25, comprising using an elevated amount of calcium for precipitating theliposome and the siRNA.
 32. The method of claim 25, comprising the stepof extruding the liposome an the siRNA prior to precipitation.
 33. Themethod of claim 25, wherein the siRNA-liposome is prepared by adding achelating agent to a cochleate to form a liposome in the presence ofsiRNA. 34-63. (canceled)